Compounds and methods for inhibiting nhe-mediated antiport in the treatment of disorders associated with fluid retention or salt overload and gastrointestinal tract disorders

ABSTRACT

The present disclosure is directed to compounds and methods for the treatment of disorders associated with fluid retention or salt overload, such as heart failure (in particular, congestive heart failure), chronic kidney disease, end-stage renal disease, liver disease, and peroxisome proliferator-activated receptor (PPAR) gamma agonist-induced fluid retention. The present disclosure is also directed to compounds and methods for the treatment of hypertension. The present disclosure is also directed to compounds and methods for the treatment of gastrointestinal tract disorders, including the treatment or reduction of pain associated with gastrointestinal tract disorders. The methods generally comprise administering to a mammal in need thereof a pharmaceutically effective amount of a compound, or a pharmaceutical composition comprising such a compound, that is designed to be substantially active in the gastrointestinal (GI) tract to inhibit NHE-mediated antiport of sodium ions and hydrogen ions therein. More particularly, the method comprises administering to a mammal in need thereof a pharmaceutically effective amount of a compound, or a pharmaceutical composition comprising such a compound, that inhibits NHE-3, -2 and/or -8 mediated antiport of sodium and/or hydrogen ions in the GI tract and is designed to be substantially impermeable to the layer of epithelial cells, or more specifically the epithelium of the GI tract. As a result of the compound being substantially impermeable, it is not absorbed and is thus essentially systemically non-bioavailable, so as to limit the exposure of other internal organs (e.g., liver, heart, brain, etc.) thereto. The present disclosure is still further directed to a method wherein a mammal is administered such a compound with a fluid-absorbing polymer, such that the combination acts as described above and further provides the ability to sequester fluid and/or salt present in the GI tract.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. patentapplication Ser. No. 15/402,211, filed Jan. 9, 2017, which is acontinuation-in-part of U.S. patent application Ser. No. 15/195,584,filed Jun. 28, 2016, which is a Continuation of U.S. patent applicationSer. No. 14/592,200, now U.S. Pat. No. 9,408,840, issued Aug. 9, 2016,which is a divisional application of U.S. patent application Ser. No.13/172,394, filed Sep. 24, 2013, now U.S. Pat. No. 8,541,448, issuedSep. 24, 2013, which is a Continuation application under 35 U.S.C. § 371of International Application No. PCT/US2009/069852, filed Dec. 30, 2009claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional PatentApplication No. 61/141,853, filed Dec. 31, 2008. U.S. Provisional PatentApplication No. 61/169,509, filed Apr. 15, 2009, and U.S. ProvisionalPatent Application No. 61/237,842, filed Aug. 28, 2009, whichapplications are incorporated herein by reference in their entireties.

FIELD OF INVENTION

The present disclosure is directed to compounds that are substantiallyactive in the gastrointestinal tract to inhibit NHE-mediated antiport ofsodium ions and hydrogen ions, and the use of such compounds in thetreatment of disorders associated with fluid retention or salt overloadand in the treatment of gastrointestinal tract disorders, including thetreatment or reduction of pain associated with a gastrointestinal tractdisorder.

DESCRIPTION OF THE RELATED ART

Disorders Associated with Fluid Retention and Salt Overload

According to the American Heart Association, more than 5 millionAmericans have suffered from heart failure, and an estimated 550,000cases of congestive heart failure (CHF) occur each year (Schocken, D. D.et al., Prevention of heart failure: a scientific statement from theAmerican Heart Association Councils on Epidemiology and Prevention.Clinical Cardiology, Cardiovascular Nursing. and High Blood PressureResearch; Quality of Care and Outcomes Research InterdisciplinaryWorking Group; and Functional Genomics and Translational BiologyInterdisciplinary Working Group: Circulation, v. 117, no. 19, p.2544-2565 (2008)). The clinical syndrome of congestive heart failureoccurs when cardiac dysfunction prevents adequate perfusion ofperipheral tissues. The most common form of heart failure leading to CHFis systolic heart failure, caused by contractile failure of themyocardium. A main cause of CHF is due to ischemic coronary arterydisease, with or without infarction. Long standing hypertension,particularly when it is poorly controlled, may lead to CHF.

In patients with CHF, neurohumoral compensatory mechanisms (i.e., thesympathetic nervous system and the renin-angiotensin system) areactivated in an effort to maintain normal circulation. Therenin-angiotensin system is activated in response to decreased cardiacoutput, causing increased levels of plasma renin, angiotensin II, andaldosterone. As blood volume increases in the heart, cardiac outputincreases proportionally, to a point where the heart is unable to dilatefurther. In the failing heart, contractility is reduced, so the heartoperates at higher volumes and higher filling pressures to maintainoutput. Filling pressures may eventually increase to a level that causestransudation of fluid into the lungs and congestive symptoms (e.g.,edema, shortness of breath). All of these symptoms are related to fluidvolume and salt retention, and this chronic fluid and salt overloadfurther contribute to disease progression.

Compliance with the medication regimen and with dietary sodiumrestrictions is a critical component of self-management for patientswith heart failure and may lengthen life, reduce hospitalizations andimprove quality of life. Physicians often recommend keeping salt intakebelow 2.3 g per day and no more than 2 g per day for people with heartfailure. Most people eat considerably more than this, so it is likelythat a person with congestive heart failure will need to find ways toreduce dietary salt.

A number of drug therapies currently exist for patients suffering fromCHF. For example, diuretics may be used or administered to relievecongestion by decreasing volume and, consequently, filling pressures tobelow those that cause pulmonary edema. By counteracting the volumeincrease, diuretics reduce cardiac output; however, fatigue anddizziness may replace CHF symptoms. Among the classes or types ofdiuretics currently being used is thiazides. Thiazides inhibit NaCltransport in the kidney, thereby preventing reabsorption of Na in thecortical diluting segment at the ending portion of the loop of Henle andthe proximal portion of the distal convoluted tubule. However, thesedrugs are not effective when the glomerular filtration rate (GFR) isless than 30 ml/min. Additionally, thiazides, as well as otherdiuretics, may cause hypokalemia. Also among the classes or types ofdiuretics currently being used is loop diuretics (e.g., furosemide).These are the most potent diuretics and are particularly effective intreating pulmonary edema. Loop diuretics inhibit the NaKCl transportsystem, thus preventing reabsorption of Na in the loop of Henle.

Patients that have persistent edema despite receiving high doses ofdiuretics may be or become diuretic-resistant. Diuretic resistance maybe caused by poor availability of the drug. In patients with renalfailure, which has a high occurrence in the CHF population, endogenousacids compete with loop diuretics such as furosemide for the organicacid secretory pathway in the tubular lumen of the nephron. Higherdoses, or continuous infusion, are therefore needed to achieve entranceof an adequate amount of drug into the nephron. However, recentmeta-analysis have raised awareness about the long-term risk of chronicuse of diuretics in the treatment of CHF. For instance, in a recentstudy (Ahmed et al., Int J Cardiol. 2008 Apr. 10; 125(2): 246-253) itwas shown that chronic diuretic use was associated with significantlyincreased mortality and hospitalization in ambulatory older adults withheart failure receiving angiotensin converting enzyme inhibitor anddiuretics.

Angiotensin-converting enzyme (“ACE”) inhibitors are an example ofanother drug therapy that may be used to treat congestive heart failure.ACE inhibitors cause vasodilatation by blocking therenin-angiotensin-aldosterone system. Abnormally low cardiac output maycause the renal system to respond by releasing renin, which thenconverts angiotensinogen into angiotensin I. ACE converts angiotensin Iinto angiotensin II. Angiotensin II stimulates the thirst centers in thehypothalamus and causes vasoconstriction, thus increasing blood pressureand venous return. Angiotensin II also causes aldosterone to bereleased, causing reabsorption of Na and concomitant passivereabsorption of fluid, which in turn causes the blood volume toincrease. ACE inhibitors block this compensatory system and improvecardiac performance by decreasing systemic and pulmonary vascularresistance. ACE inhibitors have shown survival benefit andconventionally have been a treatment of choice for CHF. However, sinceACE inhibitors lower aldosterone, the K-secreting hormone, one of theside-effects of their use is hyperkalemia. In addition, ACE inhibitorshave been show to lead to acute renal failure in certain categories ofCHF patients. (See, e.g., C. S. Cruz et al., “Incidence and Predictorsof Development of Acute Renal Failure Related to the Treatment ofCongestive Heart Failure with ACE Inhibitors, Nephron Clin. Pract., v.105, no. 2, pp c77-c83 (2007)).

Patients with end stage renal disease (“ESRD”). i.e., stage 5 chronickidney failure, must undergo hemodialysis three times per week. Thequasi-absence of renal function and ability to eliminate salt and fluidresults in large fluctuations in body weight as fluid and salt build upin the body (sodium/volume overload). The fluid overload ischaracterized as interdialytic weight gain. High fluid overload is alsoworsened by heart dysfunction, specifically CHF. Dialysis is used toremove uremic toxins and also adjust salt and fluid homeostasis.However, symptomatic intradialytic hypotension (SIH) may occur whenpatients are over-dialyzed. SIH is exhibited in about 15% to 25% of theESRD population (Davenport, A., C. Cox, and R Thuraisingham. Bloodpressure control and symptomatic intradialytic hypotension in diabetichaemodialysis patients: a cross-sectional survey; Nephron Clin. Pract.,v. 109, no. 2, p. c65-c71 (2008)). Like in hypertensive and CHFpatients, dietary restrictions of salt and fluid are highly recommendedbut poorly followed because of the poor palatability of low-salt food

The cause of primary or “essential” hypertension is elusive. However,several observations point to the kidney as a primary factor. Thestrongest data for excess salt intake and elevated blood pressure comefrom INTERSALT, a cross-sectional study of greater than 10,000participants. For individuals, a significant, positive, independentlinear relation between 24-hour sodium excretion and systolic bloodpressure was found. Higher individual 24-hour urinary sodium excretionswere found to be associated with higher systolic/diastolic bloodpressure on average, by 6-3/3-0 mm Hg. Primary hypertension is a typicalexample of a complex, multifactorial, and polygenic trait. All thesemonogenic hypertensive syndromes are virtually confined to mutated genesinvolving gain of function of various components of therenin-angiotensin-aldosterone system, resulting in excessive renalsodium retention. In a broad sense, these syndromes are characterized byincreased renal sodium reabsorption arising through either primarydefects in sodium transport systems or stimulation of mineralocorticoidreceptor activity (Altun, B., and M. Arici, 2006, Salt and bloodpressure: time to challenge; Cardiology, v. 105, no. 1, p. 9-16 (2006)).A much larger number of controlled studies have been performed onhypertensive subjects during the last three decades to determine whethersodium reduction will reduce established high blood pressure.Meta-analyses of these studies have clearly shown a large decrease inblood pressure in hypertensive patients.

In end stage liver disease (ESLD), accumulation of fluid as ascites,edema or pleural effusion due to cirrhosis is common and results from aderangement in the extracellular fluid volume regulatory mechanisms.Fluid retention is the most frequent complication of ESLD and occurs inabout 50% of patients within 10 years of the diagnosis of cirrhosis.This complication significantly impairs the quality of life of cirrhoticpatients and is also associated with poor prognosis. The one-year andfive-year survival rate is 85% and 56%, respectively (Kashani et al.,Fluid retention in cirrhosis: pathophysiology and management; QJM. v.101, no. 2, p. 71-85 (2008)). The most acceptable theories postulatethat the initial event in ascites formation in the cirrhotic patient issinusoidal hypertension. Portal hypertension due to an increase insinusoidal pressure activates vasodilatory mechanisms. In advancedstages of cirrhosis, arteriolar vasodilation causes underfilling ofsystemic arterial vascular space. This event, through a decrease ineffective blood volume, leads to a drop in arterial pressure.Consequently, baroreceptor-mediated activation of renin-angiotensinaldosterone system, sympathetic nervous system and nonosmotic release ofantidiuretic hormone occur to restore the normal blood homeostasis.These events cause further retention of renal sodium and fluid.Splanchnic vasodilation increases splanchnic lymph production, exceedingthe lymph transportation system capacity, and leads to lymph leakageinto the peritoneal cavity. Persistent renal sodium and fluid retention,alongside increased splanchnic vascular permeability in addition tolymph leakage into the peritoneal cavity, play a major role in asustained ascites formation.

Thiazolidinediones (TZD's), such as rosiglitazone, are peroxisomeproliferator-activated receptor (PPAR) gamma agonist agents used for thetreatment of type-2 diabetes and are widely prescribed. Unfortunately,fluid retention has emerged as the most common and serious side-effectof TZD's and has become the most frequent cause of discontinuation oftherapy. The incidence of TZD-induced fluid retention ranges from 7% inmonotherapy and to as high as 15% when combined with insulin (Yan, T.,Soodvilai, S., PPAR Research volume 2008, article ID 943614). Themechanisms for such side-effects are not fully understood but may berelated in Na and fluid re-absorption in the kidney. However TZD-inducedfluid retention is resistant to loop diuretics or thiazide diuretics,and combination of peroxisome proliferator-activated receptor (PPAR)alpha with PPAR gamma agonists, which were proposed to reduce such fluidoverload, are associated with major adverse cardiovascular events.

In view of the foregoing, it is recognized that salt and fluidaccumulation contribute to the morbidity and mortality of many diseases,including heart failure (in particular, congestive heart failure),chronic kidney disease, end-stage renal disease, liver disease and thelike. It is also accepted that salt and fluid accumulation are riskfactors for hypertension. Accordingly, there is a clear need for amedicament that, when administered to a patient in need, would result ina reduction in sodium retention, fluid retention, or preferably both.Such a medicament would more preferably also not involve or otherwiseimpair renal mechanisms of fluid/Na homeostasis.

One option to consider for treating excessive fluid overload is toinduce diarrhea. Diarrhea may be triggered by several agents including,for example, laxatives such as sorbitol, polyethyleneglycol, bisacodyland phenolphthaleine. Sorbitol and polyethyleneglycol triggers osmoticdiarrhea with low levels of secreted electrolytes; thus, their utilityin removing sodium salt from the GI tract is limited. The mechanism ofaction of phenolphthalein is not clearly established, but is thought tobe caused by inhibition of the Na/K ATPase and the Cl/HCO₃ anionexchanger and stimulation of electrogenic anion secretion (see, e.g.,Eherer, A. J., C. A. Santa Ana, J. Porter. and J. S. Fordtran, 1993,Gastroenterology, v. 104, no. 4, p. 1007-1012). However, some laxatives,such as phenolphthalein, are not viable options for the chronictreatment of fluid overload, due to the potential risk ofcarcinogenicity in humans. Furthermore, laxatives may not be usedchronically, as they have been shown to be an irritant and cause mucosaldamage. Accordingly, it should also be recognized that the induction ofchronic diarrhea as part of an effort to control salt and fluid overloadwould be an undesired treatment modality for most patients. Anymedicament utilizing the GI tract for this purpose would therefore needto control diarrhea in order to be of practical benefit.

One approach for the treatment of mild diarrhea is the administration ofa fluid-absorbing polymer, such as the natural plant fiber psyllium.Polymeric materials, and more specifically hydrogel polymers, may alsobe used for the removal of fluid from the gastrointestinal (GI) tract.The use of such polymers is described in, for example, U.S. Pat. Nos.4,470,975 and 6,908,609, the entire contents of which are incorporatedherein by reference for all relevant and consistent purposes. However,for such polymers to effectively remove significant quantities of fluid,they must desirably resist the static and osmotic pressure rangeexisting in the GI tract. Many mammals, including humans, make a softfeces with a water content of about 70%, and do so by transporting fluidagainst the high hydraulic resistance imposed by the fecal mass. Severalstudies show that the pressure required to dehydrate feces from about80% to about 60% is between about 500 kPa and about 1000 kPa (i.e.,about 5 to about 10 atm). (See, e.g., McKie. A. T., W. Powrie, and R. J.Naftalin, 1990, Am J Phvsiol, v. 258, no. 3 Pt 1, p. G391-G394;Bleakman, D., and R. J. Naftalin, 1990, Am J Physiol, v. 258, no. 3 Pt1, p. G377-G390; Zammit, P. S., M. Mendizabal, and R. J. Naftalin, 1994,J Physiol, v. 477 (Pt 3), p. 539-548.) However, the static pressuremeasured intraluminally is usually between about 6 kPa and about 15 kPa.The rather high pressure needed to dehydrate feces is essentially due toan osmotic process and not a mechanical process produced by muscularforces. The osmotic pressure arises from the active transport of saltacross the colonic mucosa that ultimately produces a hypertonic fluidabsorption. The osmotic gradient produced drives fluid from the lumen tothe serosal side of the mucosa. Fluid-absorbing polymers, such as thosedescribed in for example U.S. Pat. Nos. 4,470,975 and 6,908,609, may notbe able to sustain such pressure. Such polymers may collapse in a normalcolon where the salt absorption process is intact, hence removing amodest quantity of fluid and thereby salt.

Synthetic polymers that bind sodium have also been described. Forexample, ion-exchange polymeric resins, such as Dowex-type cationexchange resins, have been known since about the 1950's. However, withthe exception of Kayexalate™ (or Kionex™), which is a polystyrenesulfonate salt approved for the treatment of hyperkalemia, cationexchange resins have very limited use as drugs, due at least in part totheir limited capacity and poor cation binding selectivity.Additionally, during the ion-exchange process, the resins may release astochiometric amount of exogenous cations (e.g., H, K, Ca), which may inturn potentially cause acidosis (H), hyperkalemia (K) or contribute tovascular calcification (Ca). Such resins may also cause constipation.

Gastrointestinal Tract Disorders

Constipation is characterized by infrequent and difficult passage ofstool and becomes chronic when a patient suffers specified symptoms forover 12 non-consecutive weeks within a 12-month period. Chronicconstipation is idiopathic if it is not caused by other diseases or byuse of medications. An evidence-based approach to the management ofchronic constipation in North America (Brandt et al., 2005, Am. J.Gastroenterol. 100(Suppl. 1):S5-S21) revealed that prevalence isapproximately 15% of the general population. Constipation is reportedmore commonly in women, the elderly, non-whites, and individuals fromlower socioeconomic groups.

Irritable bowel syndrome (IBS) is a common GI disorder associated withalterations in motility, secretion and visceral sensation. A range ofclinical symptoms characterizes this disorder, including stool frequencyand form, abdominal pain and bloating. The recognition of clinicalsymptoms of IBS are yet to be defined, but it is now common to refer todiarrhea-predominant IBS (D-IBS) and constipation-predominant IBS(C-IBS), wherein D-IBS is defined as continuous passage of loose orwatery stools and C-IBS as a group of functional disorders which presentas difficult, infrequent or seemingly incomplete defecation. Thepathophysiology of IBS is not fully understood, and a number ofmechanisms have been suggested. Visceral hypersensitivity is oftenconsidered to play a major etiologic role and has been proposed to be abiological marker even useful to discriminate IBS from other causes ofabdominal pain. In a recent clinical study (Posserud, I. et al.Gastroenterology, 2007; 133:1113-1123) IBS patients were submitted to avisceral sensitivity test (Balloon distention) and compared with healthysubjects. It revealed that 61% of the IBS patients had an alteredvisceral perception as measured by pain and discomfort threshold. Otherreviews have documented the role of visceral hypersensitivity inabdominal pain symptomatic of various gastrointestinal tract disorders(Akbar, A, et al, Aliment. Pharmaco. Ther., 2009, 30, 423-435; Bueno etal., Neurogastroenterol Motility (2007) 19 (suppl. 1), 89-119). Colonicand rectal distention have been widely used as a tool to assess visceralsensitivity in animal and human studies. The type of stress used toinduce visceral sensitivity varies upon the models (see for instanceEutamen, H Neurogastroenterol Motil. 2009 Aug. 25. [Epub ahead ofprint]), however stress such as Partial restraint stress (PRS) is arelatively mild, non-ulcerogenic model that is considered morerepresentative of the IBS setting.

Constipation is commonly found in the geriatric population, particularlypatients with osteoporosis who have to take calcium supplements. Calciumsupplements have shown to be beneficial in ostoporotic patients torestore bone density but compliance is poor because of calcium-inducedconstipation effects.

Opioid-induced constipation (OIC) (also referred to as opioid-inducedbowel dysfunction or opioid bowel dysfuntion (OBD)) is a common adverseeffect associated with opioid therapy. OIC is commonly described asconstipation; however, it is a constellation of adverse gastrointestinal(GI) effects, which also includes abdominal cramping, bloating, andgastroesophageal reflux. Patients with cancer may have disease-relatedconstipation, which is usually worsened by opioid therapy. However. OICis not limited to cancer patients. A recent survey of patients takingopioid therapy for pain of non-cancer origin found that approximately40% of patients experienced constipation related to opioid therapy (<3complete bowel movements per week) compared with 7.6% in a controlgroup. Of subjects who required laxative therapy, only 46% ofopioid-treated patients (control subjects, 84%) reported achieving thedesired treatment results >50% of the time (Pappagallo, 2001, Am. J.Surg. 182(5A Suppl.): 11 S-18S).

Some patients suffering from chronic idiopathic constipation can besuccessfully treated with lifestyle modification, dietary changes andincreased fluid and fiber intake, and these treatments are generallytried first. For patients who fail to respond to these approaches,physicians typically recommend laxatives, most of which are availableover-the-counter. Use of laxatives provided over-the-counter is judgedinefficient by about half of the patients (Johanson and Kralstein, 2007,Aliment. Pharmacol. Ther. 25(5):599-608). Other therapeutic optionscurrently prescribed or in clinical development for the treatment of IBSand chronic constipation including OIC are described in, for example:Chang et al., 2006, Curr. Teat. Options Gastroenterol. 9(4):314-323;Gershon and Tack, 2007, Gastroenterology 132(1):397-414; and, Hammerleand Surawicz, 2008, World J. Gastroenterol. 14(17):2639-2649. Suchtreatments include but are not limited to serotonin receptor ligands,chloride channel activators, opioid receptor antagonists,guanylate-cyclase receptor agonists and nucleotide P2Y(2) receptoragonists. Many of these treatment options are inadequate, as they may behabit forming, ineffective in some patients, may cause long term adverseeffects, or otherwise are less than optimal.

Na⁺/H⁺ Exchanger (NHE) Inhibitors

A major function of the GI tract is to maintain water/Na homeostasis byabsorbing virtually all water and Na to which the GI tract is exposed.The epithelial layer covering the apical surface of the mammalian colonis a typical electrolyte-transporting epithelium, which is able to movelarge quantities of salt and water in both directions across the mucosa.For example, each day the GI tract processes about 9 liters of fluid andabout 800 meq of Na. (See, e.g., Zachos et al., Molecular physiology ofintestinal Na+/H+ exchange; Annu. Rev. Physiol., v. 67, p. 411-443(2005).) Only about 1.5 liters of this fluid and about 150 meq of thissodium originates from ingestion; rather, the majority of the fluid(e.g., about 7.5 liters) and sodium (about 650 meq) is secreted via theGI organs as part of digestion. The GI tract therefore represents aviable target for modulating systemic sodium and fluid levels.

Many reviews have been published on the physiology and secretory and/orabsorption mechanisms of the GI tract (see, e.g., Kunzelmann et al.,Electrolyte transport in the mammalian colon: mechanisms andimplications for disease; Physiol. Rev., v. 82, no. 1, p. 245-289(2002); Geibel, J. P.; Secretion and absorption by colonic crypts; Annu.Rev. Physiol. v. 67, p. 471-490 (2005): Zachos et al., supra; Kiela, P.R. et al., Apical NA+/H+ exchangers in the mammalian gastrointestinaltract; J. Physiol. Pharmacol., v. 57 Suppl. 7, p. 51-79 (2006)). The twomain mechanisms of Na absorption are electroneutral and electrogenictransport. Electroneutral transport is essentially due to the Na⁺/H⁺antiport NHE (e.g., NHE-3) and is responsible for the bulk of Naabsorption. Electrogenic transport is provided by the epithelium sodiumchannel (“ENaC”). Electroneutral transport is located primarily in theileal segment and proximal colon and electrogenic transport is locatedin the distal colon.

Plasma membrane NHEs contribute to maintenance of intracellular pH andvolume, transcellular absorption of NaCl and NaHCO₃, and fluid balancecarried out by epithelial cells, especially in the kidney, intestine,gallbladder, and salivary glands, as well as regulation of systemic pH.There exists a body of literature devoted to the role and clinicalintervention on systemic NHEs to treat disorders related to ischemia andreperfusion for cardioprotection or renal protection. Nine isoforms ofNHEs have been identified (Kiela. P. R., et al.; Apical Na+/H+exchangers in the mammalian gastrointestinal tract; J. Physiol.Pharmacol., v. 57 Suppl 7, p. 51-79 (2006)), of which NHE-2, NHE-3 andNHE-8 are expressed on the apical side of the GI tract, with NHE-3providing a larger contribution to transport. Another, yet to beidentified, Cl-dependant NHE has been identified in the crypt of ratcells. In addition, much research has been devoted to identifyinginhibitors of NHEs. The primary targets of such research have been NHE-1and NHE-3. Small molecule NHE inhibitors are, for example, described in:U.S. Pat. Nos. 5,866,610; 6,399,824; 6,911,453; 6,703,405; 6,005,010;6,736,705; 6,887,870; 6,737,423; 7,326,705; 5,824,691 (WO 94/026709);U.S. Pat. No. 6,399,824 (WO 02/024637); U.S. Pat. Pub. Nos. 2004/0039001(WO 02/020496); 2005/0020612 (WO 03/055490); 2004/0113396 (WO03/051866): 2005/0020612; 2005/0054705; 2008/0194621; 2007/0225323;2004/0039001; 2004/0224965; 2005/0113396; 2007/0135383; 2007/0135385;2005/0244367; 2007/0270414; International Publication Nos. WO 01/072742;WO 01021582 (CA2387529); WO 97/024113 (CA02241531) and European Pat. No.EP0744397 (CA2177007); all of which are incorporated herein by referencein their entirety for all relevant and consistent purposes. However,to-date, such research has failed to develop or recognize the value orimportance of NHE inhibitors that are not absorbed (i.e., not systemic)and target the gastrointestinal tract. Such inhibitors could be utilizedin the treatment of disorders associated with fluid retention and saltoverload and in the treatment of GI tract disorders, including thetreatment or reduction of pain associated with a gastrointestinal tractdisorder. Such inhibitors would be particular advantageous because theycould be delivered with reduced fear of systemic on-target or off-targeteffects (e.g., little or no risk of renal involvement or other systemiceffects.

Accordingly, while progress has been made in the foregoing fields, thereremains a need in the art for novel compounds for use in the disordersassociated with fluid retention and salt overload and in the treatmentof gastrointestinal tract disorders, including the treatment orreduction of pain associated with a gastrointestinal tract disorder. Thepresent invention fulfills this need and provides further relatedadvantages.

BRIEF SUMMARY OF THE INVENTION

In brief, the present invention is directed to compounds that aresubstantially active in the gastrointestinal tract to inhibitNHE-mediated antiport of sodium ions and hydrogen ions, and the use ofsuch compounds in the treatment of disorders associated with fluidretention and salt overload and in the treatment of gastrointestinaltract disorders, including the treatment or reduction of pain associatedwith a gastrointestinal tract disorder.

In one embodiment, a compound is provided having: (i) a topologicalPolar Surface Area (tPSA) of at least about 200 Å² and a molecularweight of at least about 710 Daltons in the non-salt form; or (ii) atPSA of at least about 270 Å², wherein the compound is substantiallyactive in the gastrointestinal tract to inhibit NHE-mediated antiport ofsodium ions and hydrogen ions therein upon administration to a patientin need thereof.

In further embodiments, the compound has a molecular weight of at leastabout 500 Da, at least about 1000 Da, at least about 2500 Da, or atleast about about 5000 Da.

In further embodiments, the compound has a tPSA of at least about 250Å², at least about 270 Å², at least about 300 Å², at least about 350 Å²,at least about 400 Å², or at least about 500 Å².

In further embodiments, the compound is substantially active on theapical side of the epithelium of the gastrointestinal tract to inhibitantiport of sodium ions and hydrogen ions mediated by NHE-3, NHE-2,NHE-8, or a combination thereof. In further embodiments, the compound issubstantially systemically non-bioavailable and/or substantiallyimpermeable to the epithelium of the gastrointestinal tract. In furtherembodiments, the compound is substantially active in the lowergastrointestinal tract. In further embodiments, the compound has (i) atotal number of NH and/or OH and/or other potential hydrogen bond donormoieties greater than about 5; (ii) a total number of O atoms and/or Natoms and/or other potential hydrogen bond acceptors greater than about10; and/or (iii) a Moriguchi partition coefficient greater than about10⁵ or less than about 10. In further embodiments, the compound has apermeability coefficient, P_(app), of less than about 100×10⁻⁶ cm s, orless than about 10×10⁻⁶ cm/s, or less than about 1×10⁻⁶ cm/s, or lessthan about 0.1×10⁻⁶ cm/s. In further embodiments, the compound issubstantially localized in the gastrointestinal tract or lumen. Infurther embodiments, the compound inhibits NHE irreversibly. In furtherembodiments, the compound is capable of providing a substantiallypersistent inhibitory action and wherein the compound is orallyadministered once-a-day. In further embodiments, the compound issubstantially stable under physiological conditions in thegastrointestinal tract. In further embodiments, the compound is inertwith regard to gastrointestinal flora. In further embodiments, thecompound is designed to be delivered to the lower part of thegastrointestinal tract. In further embodiments, the compound is designedto be delivered to the lower part of the gastrointestinal tract past theduodenum. In further embodiments, the compound, when administered at adose resulting in at least a 10% increase in fecal water content, has aC_(max) that is less than the IC₅₀ for NHE-3, less than about 10× theIC₅₀, or less than about 100× the IC₅₀. In further embodiments, uponadministration of the compound to a patient in need thereof, thecompound exhibits a maximum concentration detected in the serum, definedas C_(max), that is lower than the NHE inhibitory concentration IC₅₀ ofthe compound. In further embodiments, upon administration of thecompound to a patient in need thereof, greater than about 80%, greaterthan about 90% or greater than about 95% of the amount of compoundadministered is present in the patient's feces.

In further embodiments, the compound has a structure of Formula (I) or(IX):

wherein:

NHE is a NHE-inhibiting small molecule that comprises (i) a hetero-atomcontaining moiety, and (ii) a cyclic or heterocyclic scaffold or supportmoiety bound directly or indirectly thereto, the heteroatom-containingmoiety being selected from a substituted guanidinyl moiety and asubstituted heterocyclic moiety, which may optionally be fused with thescaffold or support moiety to form a fused bicyclic structure; and,

Z is a moiety having at least one site thereon for attachment to theNHE-inhibiting small molecule, the resulting NHE-Z molecule possessingoverall physicochemical properties that render it substantiallyimpermeable or substantially systemically non-bioavailable; and,

E is an integer having a value of 1 or more.

In further embodiments, the total number of freely rotatable bonds inthe NHE-Z molecule is at least about 10. In further embodiments, thetotal number hydrogen bond donors in the NHE-Z molecule is at leastabout 5. In further embodiments, the total number of hydrogen bondacceptors in the NHE-Z molecule is at least about 10. In furtherembodiments, the total number of hydrogen bond donors and hydrogen bondacceptors in the NHE-Z molecule is at least about 10. In furtherembodiments, the Log P of the NHE-Z inhibiting compound is at leastabout 5. In further embodiments, the log P of the NHE-Z inhibitingcompound is less than about 1, or less than about 0. In furtherembodiments, the scaffold is a 5-member or 6-member cyclic orheterocyclic moiety. In further embodiments, the scaffold is aromatic.

In further embodiments, the scaffold of the NHE-inhibiting smallmolecule is bound to the moiety, Z, and the compound has the structureof Formula (II):

wherein:

Z is a Core having one or more sites thereon for attachment to one ormore NHE-inhibiting small molecules, the resulting NHE-Z moleculepossessing overall physicochemical properties that render itsubstantially impermeable or substantially systemicallynon-bioavailable;

B is the heteroatom-containing moiety of the NHE-inhibiting smallmolecule, and is selected from a substituted guanidinyl moiety and asubstituted heterocyclic moiety, which may optionally be fused with theScaffold moiety to form a fused, bicyclic structure;

Scaffold is the cyclic or heterocyclic scaffold or support moiety of theNHE-inhibiting small molecule, which is bound directly or indirectly toheteroatom-containing moiety, B, and which is optionally substitutedwith one or more additionally hydrocarbyl or heterohydrocarbyl moieties;

X is a bond or a spacer moiety selected from a group consisting ofsubstituted or unsubstituted hydrocarbyl or heterohydrocarbyl moieties,and in particular substituted or unsubstituted C₁₋₇ hydrocarbyl orheterohydrocarbyl, and substituted or unsubstituted, saturated orunsaturated, cyclic or heterocyclic moieties, which links B and theScaffold; and,

D and E are integers, each independently having a value of 1 or more.

In further embodiments, the compound is an oligomer, dendrimer orpolymer, and Z is a Core moiety having two or more sites thereon forattachment to multiple NHE-inhibiting small molecules, either directlyor indirectly through a linking moiety, L, and the compound has thestructure of Formula (X):

CoreL-NHE)_(n)   (X)

wherein L is a bond or linker connecting the Core to the NHE-inhibitingsmall molecule, and n is an integer of 2 or more, and further whereineach NHE-inhibiting small molecule may be the same or differ from theothers.

In further embodiments, the NHE-inhibiting small molecule has thestructure of Formula (IV):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,

wherein:

each R₁, R₂, R₃, R₅ and R₉ are independently selected from H, halogen,—NR₇(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇, —SR₇,—O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or a bond linking the NHE-inhibiting smallmolecule to L, provided at least one is a bond linking theNHE-inhibiting small molecule to L;

R₄ is selected from H, C₁-C₇ alkyl, or a bond linking the NHE-inhibitingsmall molecule to L:

R₆ is absent or selected from H and C₁-C₇ alkyl; and

Ar1 and Ar2 independently represent an aromatic ring or a heteroaromaticring.

In further embodiments, the NHE-inhibiting small molecule has thefollowing structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,

wherein:

each R₁, R₂ and R₃ are independently selected from H, halogen,—NR₇(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇, —SR₇,—O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or a bond linking the NHE-inhibiting smallmolecule to L, provided at least one is a bond linking theNHE-inhibiting small molecule to L.

In further embodiments, the NHE-inhibiting small molecule has one of thefollowing structures:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

-   -   In further embodiments, L is a polyalkylene glycol linker. In        further embodiments, L is a polyethylene glycol linker.    -   In further embodiments, n is 2.    -   In further embodiments, the Core has the following structure:

wherein:

-   -   X is selected from the group consisting of a bond, —O—, —NH—,        —S—, C₁₋₆alkylene, —NHC(═O)—, —C(═O)NH—, —NHC(═O)NH—, —SO₂NH—,        and —NHSO₂—;    -   Y is selected from the group consisting of a bond, optionally        substituted C₁₋₈alkylene, optionally substituted aryl,        optionally substituted heteroaryl, a polyethylene glycol linker,        —(CH₂)₁₋₆O(CH₂)₁₋₆— and —(CH₂)₁₋₆NY₁(CH₂)₁₋₆—; and    -   Y₁ is selected from the group consisting of hydrogen, optionally        substituted C₁₋₈alkyl, optionally substituted aryl or optionally        substituted heteroaryl.

In further embodiments, the Core is selected from the group consistingof:

In further embodiments, the compound is an oligomer, and Z is a linkingmoiety, L, that links two or more NHE-inhibiting small moleculestogether, when the two or more NHE-inhibiting small molecules may be thesame or different, and the compound has the structure of Formula (XI):

NHEL-NHE_(m)L-NHE   (XI)

-   -   wherein L is a bond or linker connecting one NHE-inhibiting        small molecule to another, and m is 0 or an integer of 1 or        more.    -   In further embodiments, the compound is an oligomer, dendrimer        or polymer, and Z is a backbone, denoted Repeat Unit, to which        is bound multiple NHE-inhibiting moieties, and the compound has        the structure of Formula (XIIB):

-   -   wherein: L is a bond or a linking moiety; NHE is a        NHE-inhibiting small molecule; and n is a non-zero integer.

In another embodiment, a pharmaceutical composition is providedcomprising a compound as set forth above, or a stereoisomer,pharmaceutically acceptable salt or prodrug thereof, and apharmaceutically acceptable carrier, diluent or excipient.

In further embodiments, the composition further comprises afluid-absorbing polymer. In further embodiments, the fluid-absorbingpolymer is delivered directly to the colon. In further embodiments, thefluid-absorbing polymer has a fluid absorbency of at least about 15 g ofisotonic fluid per g of polymer under a static pressure of about 5 kPa.In further embodiments, the fluid-absorbing polymer has a fluidabsorbency of at least about 15 g of isotonic fluid per g of polymerunder a static pressure of about 10 kPa. In further embodiments, thefluid-absorbing polymer is characterized by a fluid absorbency of atleast about 10 g/g. In further embodiments, the fluid-absorbing polymeris characterized by a fluid absorbency of at least about 15 g/g. Infurther embodiments, the fluid-absorbing polymer is superabsorbent. Infurther embodiments, the fluid-absorbing polymer is a crosslinked,partially neutralized polyelectrolyte hydrogel. In further embodiments,the fluid-absorbing polymer is a crosslinked polyacrylate. In furtherembodiments, the fluid-absorbing polymer is a polyelectrolyte. Infurther embodiments, the fluid-absorbing polymer is calcium Carbophil.In further embodiments, the fluid-absorbing polymer is prepared by ahigh internal phase emulsion process. In further embodiments, thefluid-absorbing polymer is a foam. In further embodiments, thefluid-absorbing polymer is prepared by a aqueous free radicalpolymerization of acrylamide or a derivative thereof, a crosslinker anda free radical initiator redox system in water. In further embodiments,the fluid-absorbing polymer is a hydrogel. In further embodiments, thefluid-absorbing polymer is an N-alkyl acrylamide. In furtherembodiments, the fluid-absorbing polymer is a superporous gel. Infurther embodiments, the fluid-absorbing polymer is naturally occurring.In further embodiments, the fluid-absorbing polymer is selected from thegroup consisting of xanthan, guar, wellan, hemicelluloses,alkyl-cellulose hydro-alkyl-cellulose, carboxy-alkyl-cellulose,carrageenan, dextran, hyaluronic acid and agarose. In furtherembodiments, the fluid-absorbing polymer is psyllium. In furtherembodiments, the fluid-absorbing polymer is a polysaccharide thatincludes xylose and arabinose. In further embodiments, thefluid-absorbing polymer is a polysaccharide that includes xylose andarabinose, wherein the ratio of xylose to arabinose is at least about3:1, by weight.

In further embodiments, the composition further comprises anotherpharmaceutically active agent or compound. In further embodiments, thecomposition further comprises another pharmaceutically active agent orcompound selected from the group consisting of a diuretic, cardiacglycoside, ACE inhibitor, angiotensin-2 receptor antagonist, calciumchannel blocker, beta blocker, alpha blocker, central alpha agonist,vasodilator, blood thinner, anti-platelet agent, lipid-lowering agent,and peroxisome proliferator-activated receptor (PPAR) gamma agonistagent. In further embodiments, the diuretic is selected from the groupconsisting of a high ceiling loop diuretic, a benzothiadiazide diuretic,a potassium sparing diuretic, and a osmotic diuretic. In furtherembodiments, the composition further comprises another pharmaceuticallyactive agent or compound selected from the group consisting of ananalgesic peptide or agent. In further embodiments, the compositionfurther comprises another pharmaceutically active agent or compoundselected from the group consisting of a laxative agent selected from abulk-producing agent (e.g. psyllium husk (Metamucil)), methylcellulose(Citrucel), polycarbophil, dietary fiber, apples, stoolsofteners/surfactant (e.g., docusate, Colace, Diocto), a hydrating orosmotic agent (e.g., dibasic sodium phosphate, magnesium citrate,magnesium hydroxide (Milk of magnesia), magnesium sulfate (which isEpsom salt), monobasic sodium phosphate, sodium biphosphate), ahyperosmotic agent (e.g., glycerin suppositories, sorbitol, lactulose,and polyethylene glycol (PEG)).

In another embodiment, a method for inhibiting NHE-mediated antiport ofsodium and hydrogen ions is provided, the method comprisingadministering to a mammal in need thereof a pharmaceutically effectiveamount of a compound or pharmaceutical composition as set forth above.

In another embodiment, a method for treating a disorder associated withfluid retention or salt overload is provided, the method comprisingadministering to a mammal in need thereof a pharmaceutically effectiveamount of a compound or pharmaceutical composition as set forth above.In another embodiment, the disorder includes, but is not limited to, agastrointestinal motility disorder, irritable bowel syndrome, chronicconstipation, chronic idiopathic constipation, chronic constipationoccurring in cystic fibrosis patients, chronic constipation occurring inchronic kidney disease patients, calcium-induced constipation inosteoporotic patients, opioid-induced constipation, a functionalgastrointestinal tract disorder, gastroesophageal reflux disease,functional heartburn, dyspepsia, functional dyspepsia, non-ulcerdyspepsia, gastroparesis, chronic intestinal pseudo-obstruction. Crohn'sdisease, ulcerative colitis and related diseases referred to asinflammatory bowel syndrome, colonic pseudo-obstruction, gastric ulcers,infectious diarrhea, cancer (colorectal), “leaky gut syndrome”, cysticfibrosis gastrointestinal disease, multi-organ failure, microscopiccolitis, necrotizing enterocolitis, allergy—atopy, food allergy,infections (respiratory), acute inflammation (e.g., sepsis, systemicinflammatory response syndrome), chronic inflammation (arthritis),obesity-induced metabolic diseases (e.g., nonalcoholic steatohepatitis,Type I diabetes, Type II diabetes, cardiovascular disease), kidneydisease, diabetic kidney disease, cirrhosis, nonalcoholicsteatohepatitis, nonalcoholic fatty acid liver disease, Steatosis,primary sclerosing cholangitis, primary biliary cholangitis, portalhypertension, autoimmune disease (e.g., Type 1 diabetes, Celiac'sSecondary PTH, ankylosing spondylitis, lupus, alpecia areata, rheumatoidarthritis, polymyalgia rheumatica, fibromyalgia, chronic fatiguesyndrome, Sjogren's syndrome, vitiligo, thyroiditis, vasculitis,urticarial (hives), Raynaud's syndrome), Schizophrenia, autism spectrumdisorders, hepatic encephlopathy, small intestinal bacterial overgrowth,chronic alcoholism, and the like. In another embodiment, a method fortreating a disorder selected from the group consisting of heart failure(such as congestive heart failure), chronic kidney disease, end-stagerenal disease, liver disease, and peroxisome proliferator-activatedreceptor (PPAR) gamma agonist-induced fluid retention is provided, themethod comprising administering to a mammal in need thereof apharmaceutically effective amount of a compound or pharmaceuticalcomposition as set forth above.

In another embodiment, a method for treating hypertension is provided,the method comprising administering to a mammal in need thereof apharmaceutically effective amount of a compound or pharmaceuticalcomposition as set forth above.

In further embodiments, the method comprises administering apharmaceutically effective amount of the compound to the mammal in orderto increase the mammal's daily fecal output of sodium and/or fluid. Infurther embodiments, the method comprises administering apharmaceutically effective amount of the compound to the mammal in orderto increase the mammal's daily fecal output of sodium by at least about30 mmol, and/or fluid by at least about 200 ml. In further embodiments,the mammal's fecal output of sodium and/or fluid is increased withoutintroducing another type of cation in a stoichiometric or nearstoichiometric fashion via an ion exchange process. In furtherembodiments, the method further comprises administering to the mammal afluid-absorbing polymer to absorb fecal fluid resulting from the use ofthe compound that is substantially active in the gastrointestinal tractto inhibit NHE-mediated antiport of sodium ions and hydrogen ionstherein.

In further embodiments, the compound or composition is administered totreat hypertension. In further embodiments, the compound or compositionis administered to treat hypertension associated with dietary saltintake. In further embodiments, administration of the compound orcomposition allows the mammal to intake a more palatable diet. Infurther embodiments, the compound or composition is administered totreat fluid overload. In further embodiments, the fluid overload isassociated with congestive heart failure. In further embodiments, thefluid overload is associated with end stage renal disease. In furtherembodiments, the fluid overload is associated with peroxisomeproliferator-activated receptor (PPAR) gamma agonist therapy. In furtherembodiments, the compound or composition is administered to treat sodiumoverload. In further embodiments, the compound or composition isadministered to reduce interdialytic weight gain in ESRD patients. Infurther embodiments, the compound or composition is administered totreat edema. In further embodiments, the edema is caused bychemotherapy, pre-menstrual fluid overload or preeclampsia.

In further embodiments, the compound or composition is administered totreat gastric ulcers. In further embodiments, the compound orcomposition is administered to treat infectious diarrhea. In furtherembodiments, the compound or composition is administered to treat cancer(colorectal). In further embodiments, the compound or composition isadministered to treat “leaky gut syndrome”. In further embodiments, thecompound or composition is administered to trea cystic fibrosisgastrointestinal disease. In further embodiments, the compound orcomposition is administered to treat multi-organ failure. In furtherembodiments, the compound or composition is administered to treatmicroscopic colitis. In further embodiments, the compound or compositionis administered to treat necrotizing enterocolitis. In furtherembodiments, the compound or composition is administered to treat atopy.In further embodiments, the compound or composition is administered totreat food allergy. In further embodiments, the compound or compositionis administered to treat respiratory infections. In further embodiments,the compound or composition is administered to treat acute inflammation(e.g., sepsis, systemic inflammatory response syndrome). In furtherembodiments, the compound or composition is administered to treatchronic inflammation (e.g., arthritis). In further embodiments, thecompound or composition is administered to treat obesity-inducedmetabolic diseases (e.g., nonalcoholic steatohepatitis, Type I diabetes.Type II diabetes, cardiovascular disease). In further embodiments, thecompound or composition is administered to treat kidney disease. Infurther embodiments, the compound or composition is administered totreat diabetic kidney disease. In further embodiments, the compound orcomposition is administered to treat cirrhosis. In further embodiments,the compound or composition is administered to treat steatohepatitis. Infurther embodiments, the compound or composition is administered totreat nonalcoholic fatty acid liver disease. In further embodiments, thecompound or composition is administered to treat steatosis. In furtherembodiments, the compound or composition is administered to treatprimary sclerosing cholangitis. In further embodiments, the compound orcomposition is administered to treat primary biliary cholangitis. Infurther embodiments, the compound or composition is administered totreat portal hypertension. In further embodiments, the compound orcomposition is administered to treat autoimmunie disease (e.g., Type 1diabetes, ankylosing spondylitis, lupus, alopecia areata, rheumatoidarthritis, polymyalgia rheumatica, fibromyalgia, chronic fatiguesyndrome. Sjogren's syndrome, vitiligo, thyroiditis, vasculitis,urticarial (hives), or Raynaud's syndrome). In further embodiments, thecompound or composition is administered to treat Schizophrenia. Infurther embodiments, the compound or composition is administered totreat autism spectrum disorders. In further embodiments, the compound orcomposition is administered to treat hepatic encephlopathy. In furtherembodiments, the compound or composition is administered to treatchronic alcoholism.

In further embodiments, the compound or composition is administeredorally, by rectal suppository, or enema.

In further embodiments, the method comprises administering apharmaceutically effective amount of the compound or composition incombination with one or more additional pharmaceutically activecompounds or agents. In further embodiments, the one or more additionalpharmaceutically active compounds or agents is selected from the groupconsisting of a diuretic, cardiac glycoside, ACE inhibitor,angiotensin-2 receptor antagonist, aldosterone antagonist, calciumchannel blocker, beta blocker, alpha blocker, central alpha agonist,vasodilator, blood thinner, anti-platelet agent, lipid-lowering agent,and peroxisome proliferator-activated receptor (PPAR) gamma agonistagent. In further embodiments, the diuretic is selected from the groupconsisting of a high ceiling loop diuretic, a benzothiadiazide diuretic,a potassium sparing diuretic, and a osmotic diuretic. In furtherembodiments, the pharmaceutically effective amount of the compound orcomposition, and the one or more additional pharmaceutically activecompounds or agents, are administered as part of a single pharmaceuticalpreparation. In further embodiments, the pharmaceutically effectiveamount of the compound or composition, and the one or more additionalpharmaceutically active compounds or agents, are administered asindividual pharmaceutical preparations. In further embodiments, theindividual pharmaceutical preparation are administered sequentially. Infurther embodiments, the individual pharmaceutical preparation areadministered simultaneously.

In another embodiment, a method for treating a gastrointestinal tractdisorder is provided, the method comprising administering to a mammal inneed thereof a pharmaceutically effective amount of a compound orpharmaceutical composition as set forth above.

In further embodiments, the gastrointestinal tract disorder is agastrointestinal motility disorder. In further embodiments, thegastrointestinal tract disorder is irritable bowel syndrome. In furtherembodiments, the gastrointestinal tract disorder is chronicconstipation. In further embodiments, the gastrointestinal tractdisorder is chronic idiopathic constipation. In further embodiments, thegastrointestinal tract disorder is chronic constipation occurring incystic fibrosis patients. In further embodiments, the gastrointestinaltract disorder is opioid-induced constipation. In further embodiments,the gastrointestinal tract disorder is a functional gastrointestinaltract disorder. In further embodiments, the gastrointestinal tractdisorder is selected from the group consisting of chronic intestinalpseudo-obstruction and colonic pseudo-obstruction. In furtherembodiments, the gastrointestinal tract disorder is Crohn's disease. Infurther embodiments, the gastrointestinal tract disorder is ulcerativecolitis. In further embodiments, the gastrointestinal tract disorder isa disease referred to as inflammatory bowel disease. In furtherembodiments, the gastrointestinal tract disorder is associated withchronic kidney disease (stage 4 or 5). In further embodiments, thegastrointestinal tract disorder is associated with disturbance of pH.

In further embodiments, the gastrointestinal tract disorder tractdisorder is constipation induced by calcium supplement. In furtherembodiments, the gastrointestinal tract disorder is constipation, andthe constipation to be treated is associated with the use of atherapeutic agent. In further embodiments, the gastrointestinal tractdisorder is constipation, and the constipation to be treated isassociated with a neuropathic disorder. In further embodiments, thegastrointestinal tract disorder is constipation, and the constipation tobe treated is post-surgical constipation (postoperative ileus). Infurther embodiments, the gastrointestinal tract disorder isconstipation, and the constipation to be treated is idiopathic(functional constipation or slow transit constipation). In furtherembodiments, the gastrointestinal tract disorder is constipation, andthe constipation to be treated is associated with neuropathic, metabolicor an endocrine disorder (e.g., diabetes mellitus, renal failure,hypothyroidism, hyperthyroidism, hypocalcaemia. Multiple Sclerosis,Parkinson's disease, spinal cord lesions, neurofibromatosis, autonomicneuropathy, Chagas disease. Hirschsprung's disease or cystic fibrosis,and the like). In further embodiments, the gastrointestinal tractdisorder is constipation, and the constipation to be treated is due theuse of drugs selected from analgesics (e.g., opioids),antihypertensives, anticonvulsants, antidepressants, antispasmodics andantipsychotics.

In other embodiments, the gastrointestinal tract disorder is associatedwith gastric ulcers, infectious diarrhea, cancer (colorectal), “leakygut syndrome”, cystic fibrosis gastrointestinal disease, multi-organfailure, microscopic colitis, necrotizing enterocolitis, allergy—atopy,food allergy, infections (respiratory), acute inflammation (e.g.,sepsis, systemic inflammatory response syndrome), chronic inflammation(e.g., arthritis), obesity-induced metabolic diseases (e.g.,nonalcoholic steatohepatitis, Type I diabetes. Type II diabetes,cardiovascular disease), kidney disease, diabetic kidney disease,cirrhosis, nonalcoholic steatohepatitis, nonalcoholic fatty acid liverdisease, Steatosis, primary sclerosing cholangitis, primary biliarycholangitis, portal hypertension, autoimmune (e.g., Type I diabetes,ankylosing spondylitis, lupus, alopecia areata, rheumatoid arthritis,polymyalgia rheumatica, fibromyalgia, chronic fatigue syndrome,Sjogren's syndrome, vitiligo, thyroiditis, vasculitis, urticarial(hives), or Raynaud's syndrome). Schizophrenia, autism spectrumdisorders, hepatic encephlopathy, small intestinal bacterial overgrowth,or chronic alcoholism.

In another embodiment, a method for treating irritable bowel syndrome isprovided, the method comprising administering to a mammal in needthereof a pharmaceutically effective amount of an NHE-3 inhibitorcompound or a pharmaceutical composition comprising an NHE-3 inhibitorcompound. In further embodiments, the NHE-3 inhibitor compound or thepharmaceutical composition comprising an NHE-3 inhibitor compound is acompound or pharmaceutical composition as set forth above.

In further embodiments of the above embodiments, the compound orcomposition is administered to treat or reduce pain associated with agastrointestinal tract disorder. In further embodiments, the compound orcomposition is administered to treat or reduce visceral hypersensitivityassociated with a gastrointestinal tract disorder. In furtherembodiments, the compound or composition is administered to treat orreduce inflammation of the gastrointestinal tract. In furtherembodiments, the compound or composition is administered to reducegastrointestinal transit time.

In further embodiments, the compound or composition is administeredeither orally or by rectal suppository.

In further embodiments, the method comprises administering apharmaceutically effective amount of the compound or composition, incombination with one or more additional pharmaceutically activecompounds or agents. In further embodiments, the one or more additionalpharmaceutically active agents or compounds are an analgesic peptide oragent. In further embodiments, the one or more additionalpharmaceutically active agents or compounds are selected from the groupconsisting of a laxative agent selected from a bulk-producing agent(e.g. psyllium husk (Metamucil)), methylcellulose (Citrucel),polycarbophil, dietary fiber, apples, stool softeners/surfactant (e.g.,docusate, Colace, Diocto), a hydrating or osmotic agent (e.g., dibasicsodium phosphate, magnesium citrate, magnesium hydroxide (Milk ofmagnesia), magnesium sulfate (which is Epsom salt), monobasic sodiumphosphate, sodium biphosphate), and a hyperosmotic agent (e.g., glycerinsuppositories, sorbitol, lactulose, and polyethylene glycol (PEG)). Infurther embodiments, the pharmaceutically effective amount of thecompound or composition, and the one or more additional pharmaceuticallyactive compounds or agents, are administered as part of a singlepharmaceutical preparation. In further embodiments, the pharmaceuticallyeffective amount of the compound or composition, and the one or moreadditional pharmaceutically active compounds or agents, are administeredas individual pharmaceutical preparations. In further embodiments, theindividual pharmaceutical preparation are administered sequentially. Infurther embodiments, the individual pharmaceutical preparation areadministered simultaneously.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph that illustrates the relationship between tPSA andPermeability (Papp, as measured in the PAMPA assay) of certain examplecompounds, as further discussed in the Examples (under the subheading“2. Pharmacological Test Example 2”).

FIGS. 2A and 2B are graphs that illustrate the cecum and colon watercontent after oral administration of certain example compounds, asfurther discussed in the Examples (under the subheading “3.Pharmacological Test Example 3”).

FIGS. 3A and 3B are graphs that illustrate the dose dependent decreaseof urinary salt levels after administration of certain examplecompounds, as further discussed in the Examples (under the subheading“14. Pharmacological Test Example 14”).

FIG. 4 is a graph that illustrates a dose dependent increase in fecalwater content after administration of a certain example compound, asfurther discussed in the Examples (under the subheading “15.Pharmacological Test Example 15”).

FIGS. 5A, 5B and 5C are graphs that illustrate that supplementing thediet with Psyllium results in a slight reduction of fecal stool form,but without impacting the ability of a certain example compound toincrease fecal water content or decrease urinary sodium, as furtherdiscussed in the Examples (under the subheading “16. PharmacologicalTest Example 16”).

FIG. 6 is a graph that illustrates that inhibition of NHE-3 reduceshypersensitivity to distention, as further discussed in the Examples(under the subheading “17. Pharmacological Test Example 17”).

FIGS. 7A and 7B are graphs that illustrate that inhibition of NHE-3increases the amount of sodium excreted in feces, as further discussedin the Examples (under subheading “18. Pharmacological Test Example18”).

FIGS. 8A-8D: Depicts NHE3-independent changes in intracellular pH (pHi)modulate trans-epithelial electrical resistance in intestinal ileummonolayer cultures. Changes in pHi and trans-epithelial electricalresistance (TEER) with (A. B) nigericin and (C, D) BAM15 (3 μM) and FCCP(3 μM) compared with the known NHE3 inhibitor tenapanor and vehicle(DMSO) control in monolayer cultures. *P<0.05, **P<0.01, ***P<0.001,****P<0.0001 vs DMSO.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present disclosure, and as further detailedherein below, it has been found that the inhibition of NHE-mediatedantiport of sodium ions (Na⁺) and hydrogen ions (H⁺) in thegastrointestinal tract, and more particularly the gastrointestinalepithelia, is a powerful approach to the treatment of various disordersthat may be associated with or caused by fluid retention and/or saltoverload, and/or disorders such as heart failure (in particular,congestive heart failure), chronic kidney disease, end-stage renaldisease, liver disease, and/or peroxisome proliferator-activatedreceptor (PPAR) gamma agonist-induced fluid retention. Morespecifically, it has been found that the inhibition of the NHE-mediatedantiport of sodium ions and hydrogen ions in the GI tract increases thefecal excretion of sodium, effectively reducing systemic levels ofsodium and fluid. This, in turn, improves the clinical status of apatient suffering from, for example, CHF, ESRD/CKD and/or liver disease.It has further been found that such a treatment may optionally beenhanced by the co-administration of other beneficial compounds orcompositions, such as for example a fluid-absorbing polymer. Thefluid-absorbing polymer may optimally be chosen so that it does notblock or otherwise negatively interfere with the mechanism of action ofthe co-dosed NHE inhibitor.

Additionally, and also as further detailed herein below, it has furtherbeen found that the inhibition of NHE-mediated antiport of sodium ions(Na⁺) and hydrogen ions (H⁺) in the gastrointestinal tract, and moreparticularly the gastrointestinal epithelia, is a powerful approach tothe treatment of hypertension, that may be associated with or caused byfluid retention and/or salt overload. More specifically, it has beenfound that the inhibition of the NHE-mediated antiport of sodium ionsand hydrogen ions in the GI tract increases the fecal excretion ofsodium, effectively reducing systemic levels of sodium and fluid. This,in turn, improves the clinical status of a patient suffering fromhypertension. Such a treatment may optionally be enhanced by theco-administration of other beneficial compounds or compositions, such asfor example a fluid-absorbing polymer. The fluid-absorbing polymer mayoptimally be chosen so that it does not block or otherwise negativelyinterfere with the mechanism of action of the co-dosed NHE inhibitor,and/or hypertension.

Additionally, and also as further detailed herein below, it has furtherbeen found that the inhibition of NHE-mediated antiport of sodium ions(Na⁺) and hydrogen ions (H⁺) in the gastrointestinal tract, and moreparticularly the gastrointestinal epithelia, is a powerful approach tothe treatment of various gastrointestinal tract disorders, including thetreatment or reduction of pain associated with gastrointestinal tractdisorders, and more particularly to the restoration of appropriate fluidsecretion in the gut and the improvement of pathological conditionsencountered in constipation states. Applicants have further recognizedthat by blocking sodium ion re-absorption, the compound of the inventionrestore fluid homeostasis in the GI tract, particularly in situationswherein fluid secretion/absorption is altered in such a way that itresults in a high degree of feces dehydration, low gut motility, and/ora slow transit-time producing constipation states and GI discomfortgenerally. It has further been found that such a treatment mayoptionally be enhanced by the co-administration of other beneficialcompounds or compositions, such as for example a fluid-absorbingpolymer. The fluid-absorbing polymer may optimally be chosen so that itdoes not block or otherwise negatively interfere with the mechanism ofaction of the co-dosed NHE inhibitor.

Due to the presence of NHEs in other organs or tissues in the body, themethod of the present disclosure employs the use of compounds andcompositions that are desirably highly selective or localized, thusacting substantially in the gastrointestinal tract without exposure toother tissues or organs. In this way, any systemic effects can beminim/zed (whether they are on-target or off-target). Accordingly, it isto be noted that, as used herein, and as further detailed elsewhereherein, “substantially active in the gastrointestinal tract” generallyrefers to compounds that are substantially systemically non-bioavailableand/or substantially impermeable to the layer of epithelial cells, andmore specifically epithelium of the GI tract. It is to be further notedthat, as used herein, and as further detailed elsewhere herein.“substantially impermeable” more particularly encompasses compounds thatare impermeable to the layer of epithelial cells, and more specificallythe gastrointestinal epithelium (or epithelial layer). “Gastrointestinalepithelium” refers to the membranous tissue covering the internalsurface of the gastrointestinal tract. Accordingly, by beingsubstantially impermeable, a compound has very limited ability to betransferred across the gastrointestinal epithelium, and thus contactother internal organs (e.g., the brain, heart, liver, etc.). The typicalmechanism by which a compound can be transferred across thegastrointestinal epithelium is by either transcellular transit (asubstance travels through the cell, mediated by either passive or activetransport passing through both the apical and basolateral membranes)and/or by paracellular transit, where a substance travels between cellsof an epithelium, usually through highly restrictive structures known as“tight junctions”.

Without wishing to be bound to any particular theory, it is believedthat the NHE-inhibiting compounds (e.g., NHE-3, -2 and/or -8 inhibitors)of the present disclosure are believed to act via a distinct and uniquemechanism, to decrease paracellular permeability of the intestine. NHE3is expressed at high levels on the apical surface of thegastrointestinal tract and couples luminal Na absorption to thesecretion of intracellular protons. Inhibition of NHE3, by theNHE-inhibiting compounds (e.g., NHE-3, -2 and/or -8 inhibitors) of thepresent disclosure, results in accumulation of intracellular protons.The intracellular proton retention accompanying NHE3 inhibitionmodulates the tight junction between cells to decrease paracellularpermeability which can be measured by an increase in transepithelialelectrical resistance. Since increased paracellular and/or transcellularpermeability of the intestine is observed in many diseases including,but not limited to a gastrointestinal motility disorder, irritable bowelsyndrome, chronic constipation, chronic idiopathic constipation, chronicconstipation occurring in cystic fibrosis patients, chronic constipationoccurring in chronic kidney disease patients, calcium-inducedconstipation in osteoporotic patients, opioid-induced constipation,multiple sclerosis-induced constipation, parkinson's disease-inducedconstipation, a functional gastrointestinal tract disorder,gastroesophageal reflux disease, functional heartburn, dyspepsia,functional dyspepsia, non-ulcer dyspepsia, gastroparesis, chronicintestinal pseudo-obstruction, Crohn's disease, ulcerative colitis andrelated diseases referred to as inflammatory bowel disease, colonicpseudo-obstruction, gastric ulcers, infectious diarrhea, cancer(colorectal). “leaky gut syndrome”, cystic fibrosis gastrointestinaldisease, multi-organ failure, microscopic colitis, necrotizingenterocolitis, allergy—atopy, food allergy, infections (respiratory),acute inflammation (e.g., sepsis, systemic inflammatory responsesyndrome), chronic inflammation (arthritis), obesity-induced metabolicdiseases (e.g., nonalcoholic steatohepatitis, Type I diabetes, Type IIdiabetes, cardiovascular disease), kidney disease, diabetic kidneydisease, cirrhosis, nonalcoholic steatohepatitis, nonalcoholic fattyacid liver disease, Steatosis, primary sclerosing cholangitis, primarybiliary cholangitis, portal hypertension, autoimmune disease (e.g., Type1 diabetes, ankylosing spondylitis, lupus, alopecia areata, rheumatoidarthritis, polymyalgia rheumatica, fibromyalgia, chronic fatiguesyndrome, Sjogren's syndrome, vitiligo, thyroiditis, vasculitis,urticarial (hives). Raynaud's syndrome), Schizophrenia, autism spectrumdisorders, hepatic encephlopathy, small intestinal bactreial overgrowth,and chronic alcoholism, and the like it is anticipated that NHEinhibition could provide therapeutic benefit in these diseases bydecreasing paracellular and/or transcellular permeability in theintestine.

Thus in some embodiments, the present disclosure provides methods ofdecreasing paracellular permeability of the intestine. In someembodiments, the method of decreasing paracellular permeability of theintestine comprises administration of an NHE3 inhibitor. In someembodiments, the inhibition of NHE3 results in an accumulation ofintracellular protons. In some embodiments, the decrease in paracellularpermeability is due to an increase in intracellular protons independentof and without NHE3 inhibition. In other words, an increase inintracellular protons without NHE3 inhibition results in a decrease inparacelllar permeability. Thus methods of decreasing paracellularpermeability comprising increasing intracellular protons is provided. Insome embodiments, methods of treating diseases associated withparacellular permeability are provided comprising administering an agentthat increases intracellular protons at tight junctions therebydecreasing paracellular permeability and thus treating the disease. Nonlimiting examples of such diseases include, Crohn's disease, ulcerativecolitis and related diseases referred to as inflammatory bowel syndrome,colonic pseudo-obstruction, gastric ulcers, infectious diarrhea, cancer(colorectal). “leaky gut syndrome”, cystic fibrosis gastrointestinaldisease, multi-organ failure, microscopic colitis, necrotizingenterocolitis, allergy—atopy, food allergy, infections (respiratory),acute inflammation (e.g., sepsis, systemic inflammatory responsesyndrome), chronic inflammation (arthritis), obesity-induced metabolicdiseases (e.g., nonalcoholic steatohepatitis, Type I diabetes, Type IIdiabetes, cardiovascular disease), kidney disease, diabetic kidneydisease, cirrhosis, nonalcoholic steatohepatitis, nonalcoholic fattyacid liver disease, Steatosis, primary sclerosing cholangitis, primarybiliary cholangitis, portal hypertension, autoimmune disease (e.g., Type1 diabetes, ankylosing spondylitis, lupus, alopecia areata, rheumatoidarthritis, polymyalgia rheumatica, fibromyalgia, chronic fatiguesyndrome, Sjogren's syndrome, vitiligo, thyroiditis, vasculitis,urticarial (hives), Raynaud's syndrome). Schizophrenia, autism spectrumdisorders, hepatic encephlopathy, small intestinal bactreial overgrowth,and chronic alcoholism, and the like.

In some embodiments, the present disclosure provides methods ofmodulating transcellular permeability of the intestine. In someembodiments, the method of modulating transcellular permeability of theintestine comprises administration of an NHE3 inhibitor. In someembodiments, the inhibition of NHE3 results in a substance travellingthrough the cell, mediated by either passive or active transport passingthrough both the apical and basolateral membranes. Thus methods ofmodulating transcellular permeability comprising mediating eitherpassive or active transport of a substance passing through both theapical and basolateral membranes is provided. In some embodiments,methods of treating diseases associated with transcellular permeabilityare provided comprising administering an agent that mediates eitherpassive or active transport of a substance passing through both theapical and basolateral membranes of a cell, thereby modulatingtranscellular permeability and thus treating the disease. Non limitingexamples of such diseases include a gastrointestinal motility disorder,irritable bowel syndrome, chronic constipation, chronic idiopathicconstipation, chronic constipation occurring in cystic fibrosispatients, chronic constipation occurring in chronic kidney diseasepatients, calcium-induced constipation in osteoporotic patients,opioid-induced constipation, multiple sclerosis-induced constipation,parkinson's disease-induced constipation, a functional gastrointestinaltract disorder, gastroesophageal reflux disease, functional heartburn,dyspepsia, functional dyspepsia, non-ulcer dyspepsia, gastroparesis,chronic intestinal pseudo-obstruction.

The compounds of the present disclosure may therefore not be absorbed,and are thus essentially not systemically bioavailable at all (e.g.,impermeable to the gastrointestinal epithelium at all), or they show nodetectable concentration of the compound in serum. Alternatively, thecompounds may: (i) exhibit some detectable permeability to the layer ofepithelial cells, and more particularly the epithelium of the GI tract,of less than about 20% of the administered compound (e.g., less thanabout 15%, about 10%, or even about 5%, and for example greater thanabout 0.5%, or 1%), but then are rapidly cleared in the liver (i.e.,hepatic extraction) via first-pass metabolism; and/or (ii) exhibit somedetectable permeability to the layer of epithelial cells, and moreparticularly the epithelium of the GI tract, of less than about 20% ofthe administered compound (e.g., less than about 15%, about 10%, or evenabout 5%, and for example greater than about 0.5%, or 1%), but then arerapidly cleared in the kidney (i.e., renal excretion).

In this regard it is to be still further noted that, as used herein,“substantially systemically non-bioavailable” generally refers to theinability to detect a compound in the systemic circulation of an animalor human following an oral dose of the compound. For a compound to bebioavailable, it must be transferred across the gastrointestinalepithelium (that is, substantially permeable as defined above), betransported via the portal circulation to the liver, avoid substantialmetabolism in the liver, and then be transferred into systemiccirculation.

As further detailed elsewhere herein, small molecules exhibiting aninhibitory effect on NHE-mediated antiport of sodium and hydrogen ionsdescribed herein may be modified or functionalized to render them“substantially active” in the GI tract (or “substantially impermeable”to the GI tract and/or “substantially systemically non-bioavailable”from the GI tract) by, for example, ensuring that the final compoundhas: (i) a molecular weight of greater than about 500 Daltons (Da)(e.g., greater than about 1000 Da, about 2500 Da, about 5000 Da, or evenabout 10000 Da) in its non-salt form: and/or (ii) at least about 10freely rotatable bonds therein (e.g., about 10, about 15 or even about20): and/or (iii) a Moriguchi Partition Coefficient of at least about10⁵ (or log P of at least about 5), by for example increasing thehydrophobicity of the compound (e.g., inserting or installing ahydrocarbon chain of a sufficient or suitable length therein), oralternatively a Moriguchi Partition Coefficient of less than 10 (oralternatively a log P of less than about 1, or less than about 0);and/or (iv) a number of hydrogen-bond donors therein greater than about5, about 10, or about 15; and/or (v) a number of hydrogen-bond acceptorstherein greater than about 5, about 10, or about 15; and/or (vi) a totalnumber of hydrogen-bond donors and acceptors therein of greater thanabout 5, about 10, or about 15; and/or, (vii) a topological polarsurface area (tPSA) therein of greater than about 100 Å², about 120 Å²,about 130 Å², or about 140 Å², and in some instances about 150 Å², about200 Å², about 250 Å², about 270 Å², about 300 Å², about 400 Å², or evenabout 500 Å², by for example inserting or installing a sufficientlyhydrophilic functional group therein (e.g., a polyalkylene ether or apolyol or an ionizable group, such as a phosphonate, sulfonate,carboxylate, amine, quaternary amine, etc.), the hydrogen-bonddonors/acceptor groups also contributing to compound tPSA.

One or more of the above-noted methods for structurally modifying orfunctionalizing the NHE-inhibiting small molecule may be utilized inorder to prepare a compound suitable for use in the methods of thepresent disclosure, so as to render the compound substantiallyimpermeable or substantially systemically non-bioavailable; that is, oneor more of the noted exemplary physical properties may be “engineered”into the NHE-inhibiting small molecule to render the resulting compoundsubstantially impermeable or substantially systemicallynon-bioavailable, or more generally substantially active, in the GItract, while still possessing a region or moiety therein that is activeto inhibit NHE-mediated antiport of sodium ions and hydrogen ions.

Without being being held to any particular theory, the NHE-inhibitors(e.g., NHE-3, -2 and/or -8) of the instant disclosure are believed toact via a distinct and unique mechanism, causing the retention of fluidand ions in the GI tract (and stimulating fecal excretion) rather thanstimulating increased secretion of said fluid and ions. For example,lubiprostone (Amitiza® Sucampo/Takeda) is a bicyclic fatty acidprostaglandin E1 analog that activates the Type 2 Chloride Channel(ClC-2) and increases chloride-rich fluid secretion from the serosal tothe mucosal side of the GI tract (see, e.g., Pharmacological Reviews forAmitiza®, NDA package). Linaclotide (MD-1100 acetate, Microbia/ForestLabs) is a 14 amino acid peptide analogue of an endogenous hormone,guanylin, and indirectly activates the Cystic Fibrosis TransmembraneConductance Regulator (CFTR) thereby inducing fluid and electrolytesecretion into the GI (see. e.g., Li et al., J. Exp. Med., vol. 202(2005), pp. 975-986). The substantially impermeable NHE inhibitorsdescribed in the instant disclosure act to inhibit the reuptake of saltand fluid rather than promote secretion. Since the GI tract processesabout 9 liters of fluid and about 800 meq of Na each day, it isanticipated that NHE inhibition could permit the removal of substantialquantities of systemic fluid and sodium to resorb edema and resolve CHFsymptoms.

I. Substantially Impermeable or Substantially SystemicallyNon-Bioavailable NHE-Inhibiting Compounds A. General Structure

Generally speaking, the present disclosure encompasses essentially anysmall molecule, which may be monovalent or polyvalent, that is effectiveor active as a NHE inhibitor and that is substantially active in the GItract, and more particularly substantially impermeable or substantiallysystemically non-bioavalable therein, including known NHE inhibitorsthat may be modified or functionalized in accordance with the presentdisclosure to alter the physicochemical properties thereof so as torender the overall compound substantially active in the GI tract. Inparticular, however, the present disclosure encompasses monovalent orpolyvalent compounds that are effective or active as NHE-3, NHE-2 and/orNHE-8 inhibitors.

Accordingly, the compounds of the present disclosure may be generallyrepresented by Formula (I):

NHE-Z   (I)

wherein: (i) NHE represents a NHE-inhibiting small molecule, and (ii) Zrepresents a moiety having at least one site thereon for attachment toan NHE-inhibiting small molecule, the resulting NHE-Z moleculepossessing overall physicochemical properties that render itsubstantially impermeable or substantially systemicallynon-bioavailable. The NHE-inhibiting small molecule generally comprisesa heteroatom-containing moiety and a cyclic or heterocyclic scaffold orsupport moiety bound directly or indirectly thereto. In particular,examination of the structures of small molecules reported to-date to beNHE inhibitors suggest, as further illustrated herein below, that mostcomprise a cyclic or heterocyclic support or scaffold bound directly orindirectly (by, for example, an acyl moiety or a hydrocabyl orheterohydrocarbyl moiety, such as an alkyl, an alkenyl, a heteroalkyl ora heteroalkenyl moiety) to a heteroatom-containing moiety that iscapable of acting as a sodium atom or sodium ion mimic, which istypically selected from a substituted guanidinyl moiety and asubstituted heterocyclic moiety (e.g., a nitrogen-containing herocyclicmoiety). Optionally, the heteroatom-containing moiety may be fused withthe scaffold or support moiety to form a fused, bicyclic structure,and/or it may be capable of forming a positive charge at a physiologicalpH.

In this regard it is to be noted that, while the heteroatom-containingmoiety that is capable of acting as a sodium atom or ion mimic mayoptionally form a positive charge, this should not be understood orinterpreted to require that the overall compound have a net positivecharge, or only a single positively charged moiety therein. Rather, invarious embodiments, the compound may have no charged moieties, or itmay have multiple charged moieties therein (which may have positivecharges, negative charges, or a combination thereof, the compound forexample being a zwitterion). Additionally, it is to be understood thatthe overall compound may have a net neutral charge, a net positivecharge (e.g., +1, +2, +3, etc.), or a net negative charge (e.g., −1, −2,−3, etc.).

The Z moiety may be bound to essentially any position on, or within, theNHE small molecule, and in particular may be: (i) bound to the scaffoldor support moiety, (ii) bound to a position on, or within, theheteroatom-containing moiety, and/or (iii) bound to a position on, orwithin, a spacer moiety that links the scaffold to theheteroatom-containing moiety, provided that the installation of the Zmoiety does not significantly adversely impact NHE-inhibiting activity.In one particular embodiment, Z may be in the form of an oligomer,dendrimer or polymer bound to the NHE small molecule (e.g., bound forexample to the scaffold or the spacer moiety), or alternatively Z may bein the form of a linker that links multiple NHE small moleculestogether, and therefore that acts to increase: (i) the overall molecularweight and/or polar surface area of the NHE-Z molecule; and/or, (ii) thenumber of freely rotatable bonds in the NHE-Z molecule; and/or, (iii)the number of hydrogen-bond donors and/or acceptors in the NHE-Zmolecule; and/or, (iv) the Log P value of the NHE-Z molecule to a valueof at least about 5 (or alternatively less than 1, or even about 0), allas set forth herein; such that the overall NHE-inhibiting compound(i.e., the NHE-Z compound) is substantially impermeable or substantiallysystemically non-bioavailable.

The present disclosure is more particularly directed to such asubstantially impermeable or substantially systemicallynon-bioavailable, NHE-inhibiting compound, or a pharmaceutical saltthereof, wherein the compound has the structure of Formula (II):

wherein: (i) Z, as previously defined above, is a moiety bound to orincorporated in the NHE-inhibiting small molecule, such that theresulting NHE-Z molecule possesses overall physicochemical propertiesthat render it substantially impermeable or substantially systemicallynon-bioavailable; (ii) B is the heteroatom-containing moiety of theNHE-inhibiting small molecule, and in one particular embodiment isselected from a substituted guanidinyl moiety and a substitutedheterocyclic moiety, which may optionally be fused with the Scaffoldmoiety to form a fused, bicyclic structure; (iii) Scaffold is the cyclicor heterocyclic moiety to which is bound directly or indirectly thehetero-atom containing moiety (e.g., the substituted guanidinyl moietyor a substituted heterocyclic moiety), B, and which is optionallysubstituted with one or more additionally hydrocarbyl orheterohydrocarbyl moieties; (iv) X is a bond or a spacer moiety selectedfrom a group consisting of substituted or unsubstituted hydrocarbyl orheterohydrocarbyl moieties, and in particular substituted orunsubstituted C₁-C₇ hydrocarbyl or heterohydrocarbyl (e.g., C₁-C₇ alkyl,alkenyl, heteroalkyl or heteroalkenyl), and substituted orunsubstituted, saturated or unsaturated, cyclic or heterocyclic moieties(e.g., C₄-C₇ cyclic or heterocyclic moieties), which links B and theScaffold; and, (v) D and E are integers, each independently having avalue of 1, 2 or more.

In one or more particular embodiments, as further illustrated hereinbelow, B may be selected from a guanidinyl moiety or a moiety that is aguanidinyl bioisostere selected from the group consisting of substitutedcyclobutenedione, substituted imidazole, substituted thiazole,substituted oxadiazole, substituted pyrazole, or a substituted amine.More particularly, B may be selected from guanidinyl, acylguanidinyl,sulfonylguanidinyl, or a guanidine bioisostere such as acyclobutenedione, a substituted or unsubstituted 5- or 6-memberheterocycle such as substituted or unsubstituted imidazole,aminoimidazole, alkylimidizole, thiazole, oxadiazole, pyrazole,alkylthioimidazole, or other functionality that may optionally becomepositively charged or function as a sodium mimetic, including amines(e.g., tertiary amines), alkylamines, and the like, at a physiologicalpH. In one particularly preferred embodiment, B is a substitutedguanidinyl moiety or a substituted heterocyclic moiety that mayoptionally become positively charged at a physiological pH to functionas a sodium mimetic. In one exemplary embodiment, the compound of thepresent disclosure (or more particularly the pharmaceutically acceptableHCl salt thereof, as illustrated) may have the structure of Formula(III):

wherein Z may be optionally attached to any one of a number of sites onthe NHE-inhibiting small molecule, and further wherein the R₁, R₂ and R₃substituents on the aromatic rings are as detailed elsewhere herein,and/or in U.S. Pat. No. 6,399,824, the entire contents of which areincorporated herein by reference for all relevant and consistentpurposes.

In this regard it is to be noted, however, that the substantiallyimpermeable or substantially systemically non-bioavailableNHE-inhibiting compounds of the present disclosure may have a structureother than illustrated above, without departing from the scope of thepresent disclosure. For example, in various alternative embodiments, oneor both of the terminal nitrogen atoms in the guanidine moiety may besubstituted with one or more substituents, and/or the modifying orfunctionalizing moiety Z may be attached to the NHE-inhibiting compoundby means of (i) the Scaffold, (ii) the spacer X, or (iii) theheteroatom-containing moiety, B, as further illustrated generally in thestructures provided below:

In this regard it is to be further noted that, as used herein.“bioisostere” generally refers to a moiety with similar physical andchemical properties to a guanidine moiety, which in turn impartsbiological properties to that given moiety similar to, again, aguanidine moiety, in this instance. (See, for example, Ahmad, S. et al.,Aminoimidazoles as Bioisosteres of Acylguanidines: Novel, Potent,Selective and Orally Bioavailable Inhibitors of the Sodium HydrogenExchanger Isoform-1, Boorganic & Med. Chem. Lett., pp. 177-180 (2004),the entire contents of which is incorporated herein by reference for allrelevant and consistent purposes.)

As further detailed below, known NHE-inhibiting small molecules orchemotypes that may serve as suitable starting materials (formodification or functionalization, in order to render the smallmolecules substantially impermeable or substantially systemicallynon-bioavailable, and/or used in pharmaceutical preparations incombination with, for example, a fluid-absorbing polymer) may generallybe organized into a number of subsets, such as for example:

wherein: the terminal ring (or, in the case of the non-acyl guanidines,“R”), represent the scaffold or support moiety; the guanidine moiety (orthe substituted heterocycle, and more specifically the piperidine ring,in the case of the non-guanidine inhibitors) represents B; and, X is theacyl moiety, or the -A-B-acyl-moiety (or a bond in the case of thenon-acyl guanidines and the non-guanidine inhibitors). (See, e.g., Lang,H. J., “Chemistry of NHE Inhibitors” in The Sodium-Hydrogen Exchanger,Harmazyn, M., Avkiran, M. and Fliegel, L., Eds., Kluwer AcademicPublishers 2003. See also B. Masereel et al., An Overview of Inhibitorsof Na+/H+ Exchanger, European J. of Med. Chem., 38. pp. 547-554 (2003),the entire contents of which is incorporated by reference here for allrelevant and consistent purposes). Without being held to any particulartheory, it has been proposed that a guanidine group, or an acylguanidinegroup, or a charged guanidine or acylguanidine group (or, in the case ofnon-guanidine inhibitors, a heterocycle or other functional group thatcan replicate the molecular interactions of a guanidinyl functionalityincluding, but not limited to, a protonated nitrogen atom in apiperidine ring) at physiological pH may mimic a sodium ion at thebinding site of the exchanger or antiporter (See, e.g., Vigne, P.;Frelin, C.; Lazdunski, M. J. Biol. Chem. 1982, 257, 9394).

Although the heteroatom-containing moiety may be capable of forming apositive charge, this should not be understood or interpreted to requirethat the overall compound have a net positive charge, or only a singlepositively charged moiety therein, or even that theheteroatom-containing moiety therein be capable of forming a positivecharge in all instances. Rather, in various alternative embodiments, thecompound may have no charged moieties therein, or it may have multiplecharged moieties therein (which may have positive charges, negativecharges, or a combination thereof). Additionally, it is to be understoodthat the overall compound may have a net neutral charge, a net positivecharge, or a net negative charge.

In this regard it is to be noted that the U.S. patents and U.S.Published Applications cited above, or elsewhere herein, areincorporated herein by reference in their entirety, for all relevant andconsistent purposes.

In addition to the structures illustrated above, and elsewhere herein,it is to be noted that bioisosteric replacements for guanidine oracylguanidine may also be used. Potentially viable bioisosteric“guanidine replacements” identified to-date have a five- or six-memberedheterocyclic ring with donor/acceptor and pKa patterns similar to thatof guanidine or acylguanidine (see for example Ahmad, S. et al.,Aminoimidazoles as Bioisosteres of Acylguanidines: Novel, Potent,Selective and Orally Bioavailable Inhibitors of the Sodium HydrogenExchanger Isoform-1, Boorganic & Med. Chem. Lett., pp. 177-180 (2004),the entire contents of which is incorporated herein by reference for allrelevant and consistent purposes), and include those illustrated below:

The above bioisosteric embodiments (i.e., the group of structures above)correspond to “B” in the structure of Formula (II), the broken bondtherein being attached to “X” (e.g., the acyl moiety, or alternatively abond linking the bioisostere to the scaffold), with bonds to Z inFormula (III) not shown here.

It is to be noted that, in the many structures illustrated herein, allof the various linkages or bonds will not be shown in every instance.For example, in one or more of the structures illustrated above, a bondor connection between the NHE-inhibiting small molecule and themodifying or functionalizing moiety Z is not always shown. However, thisshould not be viewed in a limiting sense. Rather, it is to be understoodthat the NHE-inhibiting small molecule is bound or connected in some way(e.g., by a bond or linker of some kind) to Z, such that the resultingNHE-Z molecule is suitable for use (i.e., substantially impermeable orsubstantially systemically non-bioavailable in the GI tract).Alternatively, Z may be incorporated into the NHE-inhibiting smallmolecule, such as for example by positioning it between the guanidinemoiety and scaffold.

It is to be further noted that a number of structures are providedherein for substantially impermeable or substantially systemicallynon-bioavailable NHE-inhibiting compounds, and/or for NHE-inhibitingsmall molecules suitable for modification or functionalization inaccordance with the present disclosure so as to render themsubstantially impermeable or substantially systemicallynon-bioavailable. Due to the large number of structures, variousidentifiers (e.g., atom identifiers in a chain or ring, identifiers forsubstituents on a ring or chain, etc.) may be used more than once. Anidentifier in one structure should therefore not be assumed to have thesame meaning in a different structure, unless specifically stated (e.g.,“R₁” in one structure may or may not be the same as “R₁” in anotherstructure). Additionally, it is to be noted that, in one or more of thestructures further illustrated herein below, specific details of thestructures, including one or more of the identifiers therein, may beprovided in a cited reference, the contents of which are specificallyincorporated herein by reference for all relevant and consistentpurposes.

B. Illustrative Small Molecule Embodiments

The substantially impermeable or substantially systemicallynon-bioavailable NHE-inhibiting compounds of the present disclosure mayin general be derived or prepared from essentially any small moleculepossessing the ability to inhibit NHE activity, including smallmolecules that have already been reported or identified as inhibitingNHE activity but lack impermeability (i.e., are not substantiallyimpermeable). In one particularly preferred embodiment, the compoundsutilized in the various methods of the present disclosure are derived orprepared from small molecules that inhibit the NHE-3, -2, and/or -8isoforms. To-date, a considerable amount of work has been devoted to thestudy of small molecules exhibiting NHE-1 inhibition, while less hasbeen devoted for example to the study of small molecules exhibitingNHE-3 inhibition. Although the present disclosure is directed generallyto substantially impermeable or substantially systemicallynon-bioavailable NHE-inhibiting compounds, the substantially impermeableor substantially systemically non-bioavailable compounds exhibitingNHE-3, -2, and/or -8 inhibition are of particular interest. However,while it is envisioned that appropriate starting points may be themodification of known NHE-3, -2, and/or -8 inhibiting small molecules,small molecules identified for the inhibition of other NHE subtypes,including NHE-1, may also be of interest, and may be optim/zed forselectivity and potency for the NHE-3, -2, and/or -8 subtype antiporter.

Small molecules suitable for use (i.e., suitable for modification orfunctionalization in accordance with the present disclosure) to preparethe substantially impermeable or substantially systemicallynon-bioavailable NHE-inhibiting compounds of the present disclosureinclude those illustrated below. In this regard it is to be noted a bondor link to Z (i.e., the modification or functionalization that rendersthe small molecules substantially impermeable or substantiallysystemically non-bioavailable) is not specifically shown. As previouslynoted, the Z moiety may be attached to, or included within, the smallmolecule at essentially any site or position that does not interfere(e.g., stericly interfere) with the ability of the resulting compound toeffectively inhibit the NHE antiport of interest. More particularly, Zmay be attached to essentially any site on the NHE-inhibiting smallmolecule, Z for example displacing all or a portion of a substituentinitially or originally present thereon and as illustrated below,provided that the site of installation of the Z moiety does not have asubstantially adversely impact on the NHE-inhibiting activity thereof.In one particular embodiment, however, a bond or link extends from Z toa site on the small molecule that effectively positions the point ofattachment as far away (based, for example, on the number of interveningatoms or bonds) from the atom or atoms present in the resulting compoundthat effectively act as the sodium ion mimic (for example, the atom oratoms capable of forming a positive ion under physiological pHconditions). In a preferred embodiment, the bond or link will extendfrom Z to a site in a ring, and more preferably an aromatic ring, withinthe small molecule, which serves as the scaffold.

In view of the foregoing, in one particular embodiment, the followingsmall molecule, disclosed in U.S. Patent Application No. 2005/0054705,the entire content of which (and in particular the text of pages 1-2therein) is incorporated herein by reference for all relevant andconsistent purposes, may be suitable for use or modification inaccordance with the present disclosure (e.g., bound to or modified toinclude Z, such that the resulting NHE-Z molecule is substantiallyimpermeable or substantially systemically non-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.In one particularly preferred embodiment, R₆ and R₇ are a halogen (e.g.,Cl), R₅ is lower alkyl (e.g., CH₃), and R₁-R₄ are H, the compound havingfor example the structure:

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 1-2 therein) is incorporated herein for all relevantand consistent purposes, may be suitable for use or modification inaccordance with the present disclosure (e.g., bound to or modified toinclude Z, such that the resulting NHE-Z molecule is substantiallyimpermeable or substantially systemically non-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular page 49 therein) is incorporated herein for all relevant andconsistent purposes, may be suitable for use or modification inaccordance with the present disclosure (e.g., bound to or modified toinclude Z, such that the resulting NHE-Z molecule is substantiallyimpermeable or substantially systemically non-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 118-120 and 175-177 therein) is incorporated herein forall relevant and consistent purposes, may be suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 129-131 therein) is incorporated herein for allrelevant and consistent purposes, may be suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.(In this regard it is to be noted that the substituent Z within thestructure illustrated above is not to be confused with the moiety Zthat, in accordance with the present disclosure, is attached to theNHE-inhibiting small molecule in order effective render the resulting“NHE-Z” molecule substantially impermeable.)

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 127-129 therein) is incorporated herein for allrelevant and consistent purposes, may be suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.(In this regard it is to be noted that Z within the ring of thestructure illustrated above is not to be confused with the moiety Zthat, in accordance with the present disclosure, is attached to theNHE-inhibiting small molecule in order effective render the resulting“NHE-Z” molecule substantially impermeable.)

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 134-137 therein) is incorporated herein for allrelevant and consistent purposes, may be suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 31-32 and 137-139 therein) is incorporated herein forall relevant and consistent purposes, may be suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 37-45 therein) is incorporated herein for all relevantand consistent purposes, may be suitable for use or modification inaccordance with the present disclosure (e.g., bound to or modified toinclude Z, such that the resulting NHE-Z molecule is substantiallyimpermeable or substantially systemically non-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.(In this regard it is to be noted that Z within the ring structureillustrated above is not to be confused with the moiety Z that, inaccordance with the present disclosure, is attached to theNHE-inhibiting small molecule in order effective render the resulting“NHE-Z” molecule substantially impermeable.)

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 100-102 therein) is incorporated herein for allrelevant and consistent purposes, may be suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference(wherein, in particular, the wavy bonds indicate variable length, or avariable number of atoms, therein).

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 90-91 therein) is incorporated herein for all relevantand consistent purposes, may be suitable for use or modification inaccordance with the present disclosure (e.g., bound to or modified toinclude Z, such that the resulting NHE-Z molecule is substantiallyimpermeable or substantially systemically non-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in U.S. Pat. No. 5,900,436 (or EP 0822182 B1), the entirecontents of which (and in particular column 1, lines 10-55 therein) areincorporated herein by reference for all relevant and consistentpurposes, may be suitable for use or modification in accordance with thepresent disclosure (e.g., bound to or modified to include Z, such thatthe resulting NHE-Z molecule is substantially impermeable orsubstantially systemically non-bioavailable).

The variables in the structures are defined in the cited patents, thedetails of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 35-47 therein) is incorporated herein for all relevantand consistent purposes, may be suitable for use or modification inaccordance with the present disclosure (e.g., bound to or modified toinclude Z, such that the resulting NHE-Z molecule is substantiallyimpermeable or substantially systemically non-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 154-155 therein) is incorporated herein for allrelevant and consistent purposes, may be suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 132-133 therein) is incorporated herein for allrelevant and consistent purposes, may be suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particular embodiment, the following small molecule,disclosed in Canadian Patent Application No. 2,241,531 (or InternationalPatent Publication No. WO 97/24113), the entire content of which (and inparticular pages 58-65 AND 141-148 therein) is incorporated herein forall relevant and consistent purposes, may be suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

The variables in the structure are defined in the cited patentapplication, the details of which are incorporated herein by reference.(In this regard it is to be noted that Z within the ring structureillustrated above is not to be confused with the moiety Z that, inaccordance with the present disclosure, is attached to theNHE-inhibiting small molecule in order effective render the resulting“NHE-Z” molecule substantially impermeable.)

In yet another particular embodiment, the following small molecule,disclosed in U.S. Pat. Nos. 6,911,453 and 6,703,405, the entire contentsof which (and in particular the text of columns 1-7 and 46 of U.S. Pat.No. 6,911,453 and columns 14-15 of U.S. Pat. No. 6,703,405) areincorporated herein by reference for all relevant and consistentpurposes, may be suitable for use or modification in accordance with thepresent disclosure (e.g., bound to or modified to include Z, such thatthe resulting NHE-Z molecule is substantially impermeable orsubstantially systemically non-bioavailable).

The variables in the structure are defined in the cited patents, thedetails of which are incorporated herein by reference. A particularlypreferred small molecule falling within the above-noted structure isfurther illustrated below (see, e.g., Example 1 of the U.S. Pat. No.6,911,453 patent, the entire contents of which are specificallyincorporated herein by reference):

In yet another particular embodiment, the following small molecules,disclosed in U.S. Patent Publication Nos. 2004/0039001, 2004/0224965,2005/0113396 and 2005/0020612, the entire contents of which areincorporated herein by reference for all relevant and consistentpurposes, may be suitable for use or modification in accordance with thepresent disclosure (e.g., bound to or modified to include Z, such thatthe resulting NHE-Z molecule is substantially impermeable orsubstantially systemically non-bioavailable).

The variables in the structures are defined above and/or in one or moreof the cited patent applications, the details of which are incorporatedherein by reference, and/or as illustrated above (wherein the brokenbonds indicate a point of attachment for the Y moiety to the fusedheterocyclic ring). In particular, in various embodiments thecombination of X and Y may be as follows:

In a particularly preferred embodiment of the above-noted structure, thesmall molecule has the general structure:

wherein R₁, R₂ and R₃ may be the same or different, but are preferablydifferent, and are independently selected from H, NR′R″ (wherein R′ andR″ are independently selected from H and hydrocarbyl, such as loweralkyl, as defined elsewhere herein) and the structure:

In a more particularly preferred embodiment of the above structure, asmall molecule falling within the above-noted structure is furtherillustrated below (see, e.g., compound I1 on p. 5 of the 2005/0020612patent application, the entire contents of which are specificallyincorporated herein by reference):

In another particularly preferred embodiment, the following smallmolecule, disclosed in U.S. Pat. No. 6,399,824, the entire content ofwhich (and in particular the text of Example 1 therein) is incorporatedherein by reference for all relevant and consistent purposes, may beparticularly suitable for use or modification in accordance with thepresent disclosure (e.g., bound to or modified to include Z, such thatthe resulting NHE-Z molecule is substantially impermeable orsubstantially systemically non-bioavailable).

In the structure, R may be preferably selected from H and(CH₃)₂NCH₂CH₂—, with H being particularly preferred in variousembodiments.

In yet another particular embodiment, the following small molecule,disclosed in U.S. Pat. No. 6,005,010 (and in particular columns 1-3therein), and/or U.S. Pat. No. 6,166,002 (and in particular columns 1-3therein), the entire contents of which are incorporated herein byreference for all relevant and consistent purposes, may be suitable foruse or modification in accordance with the present disclosure (e.g.,bound to or modified to include Z, such that the resulting NHE-Zmolecule is substantially impermeable or substantially systemicallynon-bioavailable).

The variable (“R”) in the structure is defined in the cited patentapplication, the details of which are incorporated herein by reference.

In yet another particularly preferred embodiment, the following smallmolecule, disclosed in U.S. Patent Application No. 2008/0194621, theentire content of which (and in particular the text of Example 1therein) is incorporated herein by reference for all relevant andconsistent purposes, may be particularly suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

R₁ R₂ R₃

—H —H —NH2 —H —H —H

—H —H —NH2 —H —H —H —NH2

The variables (“R₁”, “R₂ and “R₃”) in the structure are as definedabove, and/or as defined in the cited patent application, the details ofwhich are incorporated herein by reference.

In yet another particularly preferred embodiment, the following smallmolecule, disclosed in U.S. Patent Application No. 2007/0225323, theentire content of which (and in particular the text of Example 36therein) is incorporated herein by reference for all relevant andconsistent purposes, may be particularly suitable for use ormodification in accordance with the present disclosure (e.g., bound toor modified to include Z, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable).

In yet another particularly preferred embodiment, the following smallmolecule, disclosed in U.S. Pat. No. 6,911,453, the entire content ofwhich (and in particular the text of Example 35 therein) is incorporatedherein by reference for all relevant and consistent purposes, may beparticularly suitable for use or modification in accordance with thepresent disclosure (e.g., bound to or modified to include Z, such thatthe resulting NHE-Z molecule is substantially impermeable orsubstantially systemically non-bioavailable).

In one particularly preferred embodiment of the present disclosure, thesmall molecule may be selected from the group consisting of:

In these structures, a bond or link (not shown) may extend, for example,between the Core and amine-substituted aromatic ring (first structure),the heterocyclic ring or the aromatic ring to which it is bound, oralternatively the chloro-substituted aromatic ring (second structure),or the difluoro-substituted aromatic ring or the sulfonamide-substitutedaromatic ring (third structure).

C. Exemplary Small Molecule Selectivity

Shown below are examples of various NHE inhibiting small molecules andtheir selectivity across the NHE-1, -2 and -3 isoforms. (See, e.g., B.Masereel et al., An Overview of Inhibitors of Na+/H+ Exchanger, EuropeanJ. of Med. Chem., 38, pp. 547-554 (2003), the entire contents of whichis incorporated by reference here for all relevant and consistentpurposes). Most of these small molecules were optim/zed as NHE-1inhibitors, and this is reflected in their selectivity with respectthereto (IC50's for subtype-1 are significantly more potent (numericallylower) than for subtype-3). However, the data in Table 1 indicates thatNHE-3 activity may be engineered into an inhibitor series originallyoptim/zed against a different isoform. For example, amiloride is a poorNHE-3 inhibitor and was inactive against this antiporter at the highestconcentration tested (IC50>100 μM); however, analogs of this compound,such as DMA and EIPA, have NHE-3 IC50's of 14 and 2.4 uM, respectively.The cinnamoylguanidine S-2120 is over 500-fold more active against NHE-1than NHE-3; however, this selectivity is reversed in regioisomer S-3226.It is thus possible to engineer NHE-3 selectivity into a chemical seriesoptim/zed for potency against another antiporter isoform; that is, theinhibitor classes exemplified in the art may be suitably modified foractivity and selectivity against NHE-3 (or alternatively NHE-2 and/orNHE-8), as well as being modified to be rendered substantiallyimpermeable or substantially systemically non-bioavailable.

TABLE 1 IC₅₀ or K_(i) (μM)^(b) Drug^(a) NHE-1 NHE-2 NHE-3 NHE-5Amiloride   1-1.6* 1.0** >100*    21    EIPA 0.01*- 0.08*- 2.4* 0.420.02** 0.5** HMA 0.013* — 2.4* 0.37 DMA 0.023* 0.25* 14*   — Cariporide0.03-3.4  4.3-62    1->100 >30   Eniporide 0.005-0.38   2-17100-460 >30   Zoniporide 0.059  12    >500*    — BMS-284640 0.009 1800     >30    3.36 T-162559 (S) 0.001  0.43 11   — T-162559 (R) 35    0.31 >30    — S-3226 3.6   80**   0.02 S-2120 0.002  0.07 1.32 *= fromrat, **= from rabbit. NA = not active ^(a)Table adapted from Masereel,B. et al., European Journal of Medicinal Chemistry, 2003, 38, 547-54.^(b)K_(i) values are in italic

As previously noted above, the NHE inhibitor small molecules disclosedherein, including those noted above, may advantageously be modified torender them substantially impermeable or substantially systemicallynon-bioavailable. The compounds as described herein are, accordingly,effectively localized in the gastrointestinal tract or lumen, and in oneparticular embodiment the colon. Since the various NHE isomforms may befound in many different internal organs (e.g., brain, heart, liver,etc.), localization of the NHE inhibitors in the intestinal lumen isdesirable in order to minim/ze or eliminate systemic effects (i.e.,prevent or significantly limit exposure of such organs to thesecompounds). Accordingly, the present disclosure provides NHE inhibitors,and in particular NHE-3, -2 and/or -8 inhibitors, that are substantiallysystemically non-bioavailable in the GI tract, and more specificallysubstantially systemically impermeable to the gut epithelium, as furtherdescribed below.

D. Preferred Embodiments

In one or more particularly preferred embodiments of the presentdisclosure, the “NHE-Z” molecule is monovalent; that is, the moleculecontains one moiety that effectively acts to inhibit NHE-mediatedantiport of sodium ions and hydrogen ions. In such embodiments, theNHE-Z molecule may be selected, for example, from one of the followingstructures of Formulas (IV), (V), (VI) or (VII):

wherein: each R₁, R₂, R₃, R₅ and R₉ are independently selected from H,halogen (e.g., Cl), —NR₇(CO)R₈, —(CO)NR₇R₈. —SO₂—NR₇R₈, —NR₇SO₂R₈,—NR₇R₈, —OR₇, —SR₇, —O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇and R₈ are independently selected from H or Z, where Z is selected fromsubstituted or unsubstituted hydrocarbyl, heterohydrocarbyl,polyalkylene glycol and polyols, where substituents thereon are selectedfrom hydroxyls, amines, amidines, carboxylates, phosphonates,sulfonates, and guanidines; R₄ is selected from H, C₁-C₇ alkyl or Z,where Z is selected from substituted or unsubstituted hydrocarbyl,heterohydrocarbyl, a polyalkylene glycol and polyols, where substituentsthereon are selected from hydroxyls, amines, amidines, carboxylates,phosphonates, sulfonates, and guanidines; R₆ is absent or selected fromH and C₁-C₇ alkyl; and, Ar1 and Ar2 independently represent an aromaticring, or alternatively a heteroaromatic ring wherein one or more of thecarbon atoms therein is replaced with a N, O or S atom;

wherein: each R₁, R₂, R₃, and R₅ are independently selected from H,—NR—(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇—, —SR₇,—O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or Z, where Z is selected from substitutedor unsubstituted hydrocarbyl, heterohydrocarbyl, polyalkylene glycol andpolyols, where substituents thereon are selected from hydroxyls, amines,amidines, carboxylates, phosphonates, sulfonates, and guanidines,optionally linked to the ring Ar1 by a heterocyclic linker; R₄ and R₁₂are independently selected from H and R₇, where R₇ is as defined above;R₁₀ and R₁₁, when presented, are independently selected from H and C₁-C₇alkyl; and, Ar1 and Ar2 independently represent an aromatic ring, oralternatively a heteroaromatic ring wherein one or more of the carbonatoms therein is replaced with a N, O or S atom;

wherein: each X is a halogen atom, which may be the same or different;R₁ is selected from —SO₂—NR₇R₈, —NR₇(CO)R₈, —(CO)NR₇R₈, —NR₇SO₂R₈,—NR₇R₈, —OR₇, —SR₇, —O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇and R₈ are independently selected from H or Z, where Z is selected fromsubstituted or unsubstituted hydrocarbyl, heterohydrocarbyl,polyalkylene glycol and polyols, where substituents thereon are selectedfrom hydroxyls, amines, amidines, carboxylates, phosphonates,sulfonates, and guanidines; R₃ is selected from H or R₇, where R₇ is asdescribed above; R₁₃ is selected from substituted or unsubstituted C₁-C₈alkyl; R₂ and R₁₂ are independently selected from H or R₇, wherein R₇ isas described above; R₁₀ and R₁₁, when present, are independentlyselected from H and C₁-C₇ alkyl; Ar1 represents an aromatic ring, oralternatively a heteroaromatic ring wherein one or more of the carbonatoms therein is replaced with a N, O or S atom; and Ar2 represents anaromatic ring, or alternatively a heteroaromatic ring wherein one ormore of the carbon atoms therein is replaced with a N, O or S atom.

In one particular embodiment for the structure of Formula (V), one ofR₁, R₂ and R₃ is linked to the ring Ar1, and/or R₅ is linked to the ringAr2, by a heterocyclic linker having the structure:

wherein R represents R₁, R₂, R₃, or R₅ bound thereto.

In another particular embodiment, the NHE-Z molecule of the presentdisclosure may have the structure of Formula (IV):

wherein: each R₁, R₂, R₃, R₅ and R₉ are independently selected from H,halogen, NR₇(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇,—SR₇, —O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or Z, where Z is selected from substitutedhydrocarbyl, heterohydrocarbyl, or polyols and/or substituted orunsubstituted polyalkylene glycol, wherein substituents thereon areselected from the group consisting of phosphinates, phosphonates,phosphonamidates, phosphates, phosphonthioates and phosphonodithioates;R₄ is selected from H or Z, where Z is substituted or unsubstitutedhydrocarbyl, heterohydrocarbyl, a polyalkylene glycol and a polyol,where substituents thereon are selected from hydroxyls, amines,amidines, carboxylates, phosphonates, sulfonates, and guanidines; R₆ isselected from —H and C₁-C₇ alkyl; and, Ar1 and Ar2 independentlyrepresent an aromatic ring, or alternatively a heteroaromatic ringwherein one or more of the carbon atoms therein is replaced with a N, Oor S atom.

Additionally, or alternatively, in one or more embodiments of thecompounds illustrated above, the compound may optionally have a tPSA ofat least about 100 Å², about 150 Å², about 200 Å², about 250 Å², about270 Å², or more and/or a molecular weight of at least about 710 Da.

II. Polyvalent Structures: Macromolecules and Oligomers A. GeneralStructure

As noted above, the compounds of the present disclosure comprise aNHE-inhibiting small molecule that has been modified or functionalizedstructurally to alter its physicochemical properties (by the attachmentor inclusion of moiety Z), and more specifically the physicochemicalproperties of the NHE-Z molecule, thus rendering it substantiallyimpermeable or substantially systemically non-bioavailable. In oneparticular embodiment, and as further detailed elsewhere herein, theNHE-Z compound may be polyvalent (i.e., an oligomer, dendrimer orpolymer moiety), wherein Z may be referred to in this embodimentgenerally as a “Core” moiety, and the NHE-inhibiting small molecule maybe bound, directly or indirectly (by means of a linking moiety) thereto,the polyvalent compounds having for example one of the following generalstructures of Formula (VIII), (IX) and (X):

wherein: Core (or Z) and NHE are as defined above; L is a bond orlinker, as further defined elsewhere herein below, and E and n are bothan integer of 2 or more. In various alternative embodiments, however,the NHE-inhibiting small molecule may be rendered substantiallyimpermeable or substantially systemically non-bioavailable by forming apolymeric structure from multiple NHE-inhibiting small molecules, whichmay be the same or different, connected or bound by a series of linkers,L, which also may be the same or different, the compound having forexample the structure of Formula (XI):

wherein: Core (or Z) and NHE are as defined above; L is a bond orlinker, as further defined elsewhere herein below, and m is 0 or aninteger of 1 or more. In this embodiment, the physicochemicalproperties, and in particular the molecular weight or polar surfacearea, of the NHE-inhibiting small molecule is modified (e.g., increased)by having a series of NHE-inhibiting small molecules linked together, inorder to render them substantially impermeable or substantiallysystemically non-bioavailable. In these or yet additional alternativeembodiments, the polyvalent compound may be in dimeric, oligomeric orpolymeric form, wherein for example Z or the Core is a backbone to whichis bound (by means of a linker, for example) multiple NHE-inhibitingsmall molecules. Such compounds may have, for example, the structures ofFormulas (XIIA) or (XIIB):

wherein: L is a linking moiety; NHE is a NHE-inhibiting small molecule,each NHE as described above and in further detail hereinafter; and n isa non-zero integer (i.e., an integer of 1 or more).

The Core moiety has one or more attachment sites to which NHE-inhibitingsmall molecules are bound, and preferably covalently bound, via a bondor linker, L. The Core moiety may, in general, be anything that servesto enable the overall compound to be substantially impermeable orsubstantially systemically non-bioavailable (e.g., an atom, a smallmolecule, etc.), but in one or more preferred embodiments is anoligomer, a dendrimer or a polymer moiety, in each case having more thanone site of attachment for L (and thus for the NHE-inhibiting smallmolecule). The combination of the Core and NHE-inhibiting small molecule(i.e., the “NHE-Z” molecule) may have physicochemical properties thatenable the overall compound to be substantially impermeable orsubstantially systemically non-bioavailable.

In this regard it is to be noted that the repeat unit in Formulas (XIIA)and (XIIB) generally encompasses repeating units of various polymericembodiments, which may optionally be produced by methods referred toherein. In each polymeric, or more general polyvalent, embodiment, it isto be noted that each repeat unit may be the same or different, and mayor may not be linked to the NHE-inhibiting small molecule by a linker,which in turn may be the same or different when present. In this regardit is to be noted that as used herein, “polyvalent” refers to a moleculethat has multiple (e.g., 2, 4, 6, 8, 10 or more) NHE-inhibiting moietiestherein.

In this regard it is to be still further noted that, as furtherillustrated elsewhere herein, certain polyvalent NHE-inhibitingcompounds of the present disclosure show unexpectedly higher potency, asmeasured by inhibition assays (as further detailed elsewhere herein) andcharacterized by the concentration of said NHE inhibitor resulting in50% inhibition (i.e., the IC₅₀ values). It has been observed thatcertain multivalent structures, represented generally by Formula (X),above, have an IC₅₀ value several fold lower in magnitude than theindividual NHE, or L-NHE, structure (which may be referred to as the“monomer” or monovalent form). For example, in one embodiment,multivalent compounds according to Formula (X) were observed to have anIC₅₀ value of at least about 5 time lower (i.e. potency about 5 timehigher) than the monomer (or monovalent) form (e.g. Examples 46 and 49).In another embodiment, multivalent compounds according to Formula (X)were observed to have an IC₅₀ value of at least about 10 time lower(i.e. potency about 10 time higher) than the monomer form (e.g. Examples87 and 88).

The above noted embodiments are further illustrated herein below. Forexample, the first representation below of an exemplary oligomercompound, wherein the various parts of the compound corresponding to thestructure of Formula (X) are identified, is intended to provide a broadcontext for the disclosure provided herein. It is to be noted that whileeach “NHE” moiety (i.e., the NHE small molecule) in the structure belowis the same, it is within the scope of this disclosure that each isindependently selected and may be the same or different. In theillustration below, the linker moiety is a polyethylene glycol (PEG)motif. PEG derivatives are advantageous due in part to their aqueoussolubility, which may help avoid hydrophobic collapse (theintramolecular interaction of hydrophobic motifs that can occur when ahydrophobic molecule is exposed to an aqueous environment (see, e.g.,Wiley, R. A.; Rich, D. H. Medical Research Reviews 1993, 13(3),327-384). The core moiety illustrated below is also advantageous becauseit provides some rigidity to the Core-(L-NHE)_(n) molecule, allowing anincrease in distance between the NHE inhibitors while minimallyincreasing rotational degrees of freedom.

In an alternative embodiment (e.g., Formula (XI), wherein m=0), thestructure may be for example:

Within the polyvalent compounds utilized for treatments according to thepresent disclosure, n and m (when m is not zero) may be independentlyselected from the range of from about 1 to about 10, more preferablyfrom about 1 to about 5, and even more preferably from about 1 to about2. In alternative embodiments, however, n and m may be independentlyselected from the range of from about 1 to about 500, preferably fromabout 1 to about 300, more preferably from about 1 to about 100, andmost preferably from about 1 to about 50. In these or other particularembodiments, n and m may both be within the range of from about 1 toabout 50, or from about 1 to about 20.

The structures provided above are illustrations of one embodiment ofcompounds utilized for administration wherein absorption is limited(i.e., the compound is rendered substantially impermeable orsubstantially systemically non-bioavailable) by means of increasing themolecular weight of the NHE-inhibiting small molecule. In an alternativeapproach, as noted elsewhere herein, the NHE-inhibiting small moleculemay be rendered substantially impermeable or substantially systemicallynon-bioavailable by means of altering, and more specifically increasing,the topological polar surface area, as further illustrated by thefollowing structures, wherein a substituted aromatic ring is bound tothe “scaffold” of the NHE-inhibition small molecule. The selection ofionizable groups such as phosphonates, sulfonates, guanidines and thelike may be particularly advantageous at preventing paracellularpermeability. Carbohydrates are also advantageous, and though uncharged,significantly increase tPSA while minimally increasing molecular weight.

It is to be noted, within one or more of the various embodimentsillustrated herein, NHE-inhibiting small molecules suitable for use(i.e., suitable for modification or functionalization, in order torender them substantially impermeable or substantially systemicallynon-bioavailable) may, in particular, be selected independently from oneor more of the small molecules described as benzoylguandines,heteroaroylguandines, “spacer-stretched” aroylguandines, non-acylguanidines and acylguanidine isosteres, above, and as discussed infurther detail hereinafter and/or to the small molecules detailed in,for example: U.S. Pat. Nos. 5,866,610; 6,399,824; 6,911,453; 6,703,405;6,005,010; 6,887,870; 6,737,423; 7,326,705; 55,824,691 (WO94/026709);U.S. Pat. No. 6,399,824 (WO02/024637); US 2004/0339001 (WO02/020496); US2005/0020612 (WO03/055490); WO01/072742; CA 2387529 (WO01021582); CA02241531 (WO97/024113); US 2005/0113396 (WO03/051866); US2005/0020612;US2005/0054705; US2008/0194621; US2007/0225323; US2004/0039001;US2004/0224965; US2005/0113396 US2007/0135383; US2007/0135385;US2005/0244367; US2007/0270414; and CA 2177007 (EP0744397), the entirecontents of which are incorporated herein by reference for all relevantand consistent purposes. Again, it is to be noted that when it is saidthat NHE-inhibiting small molecule is selected independently, it isintended that, for example, the oligomeric structures represented inFormulas (X) and (XI) above can include different structures of the NHEsmall molecules, within the same oligomer or polymer. In other words,each “NHE” within a given polyvalent embodiment may independently be thesame or different than other “NHE” moieties within the same polyvalentembodiment.

In designing and making the substantially impermeable or substantiallysystemically non-bioavailable, NHE-inhibiting compounds that may beutilized for the treatments detailed in the instant disclosure, it mayin some cases be advantageous to first determine a likely point ofattachment on a small molecule NHE inhibitor, where a core or linkermight be installed or attached before making a series of candidatemultivalent or polyvalent compounds. This may be done by one skilled inthe art via known methods by systematically installing functionalgroups, or functional groups displaying a fragment of the desired coreor linker, onto various positions of the NHE inhibitor small moleculeand then testing these adducts to determine whether the modifiedinhibitor still retains desired biological properties (e.g., NHEinhibition). An understanding of the SAR of the inhibitor also allowsthe design of cores and/or linkers that contribute positively to theactivity of the resulting compounds. For example, the SAR of an NHEinhibitor series may show that installation of an N-alkylated piperazinecontributes positively to biochemical activity (increased potency) orpharmaceutical properties (increased solubility); the piperazine moietymay then be utilized as the point of attachment for the desired core orlinker via N-alkylation. In this fashion, the resulting compound therebyretains the favorable biochemical or pharmaceutical properties of theparent small molecule. In another example, the SAR of an NHE inhibitorseries might indicate that a hydrogen bond donor is important foractivity or selectivity. Core or linker moieties may then be designed toensure this H-bond donor is retained. These cores and/or linkers may befurther designed to attenuate or potentiate the pKa of the H-bond donor,potentially allowing improvements in potency and selectivity. In anotherscenario, an aromatic ring in an inhibitor could be an importantpharmacophore, interacting with the biological target via a pi-stackingeffect or pi-cation interaction. Linker and core motifs may be similarlydesigned to be isosteric or otherwise synergize with the aromaticfeatures of the small molecule. Accordingly, once the structure-activityrelationships within a molecular series are understood, the molecules ofinterest can be broken down into key pharmacophores which act asessential molecular recognition elements. When considering theinstallation of a core or linker motif, said motifs can be designed toexploit this SAR and may be installed to be isosteric and isoelectronicwith these motifs, resulting in compounds that retain biologicalactivity but have significantly reduced permeability.

Another way the SAR of an inhibitor series can be exploited in theinstallation of core or linker groups is to understand which regions ofthe molecule are insensitive to structural changes. For example, X-rayco-crystal structures of protein-bound inhibitors can reveal thoseportions of the inhibitor that are solvent exposed and not involved inproductive interactions with the target. Such regions can also beidentified empirically when chemical modifications in these regionsresult in a “flat SAR” (i.e., modifications appear to have minimalcontribution to biochemical activity). Those skilled in the art havefrequently exploited such regions to engineer in pharmaceuticalproperties into a compound, for example, by installing motifs that mayimprove solubility or potentiate ADME properties. In the same fashion,such regions are expected to be advantageous places to install core orlinker groups to create compounds as described in the instantdisclosure. These regions are also expected to be sites for adding, forexample, highly polar functionality such as carboxylic acids, phosphonicacids, sulfonic acids, and the like in order to greatly increase tPSA.

Another aspect to be considered in the design of cores and linkersdisplaying an NHE inhibitor is the limiting or preventing of hydrophobiccollapse. Compounds with extended hydrocarbon functionalities maycollapse upon themselves in an intramolecular fashion, causing anincreased enthalpic barrier for interaction with the desired biologicaltarget. Accordingly, when designing cores and linkers, these arepreferably designed to be resistant to hydrophobic collapse. Forexample, conformational constraints such as rigid monocyclic, bicyclicor polycyclic rings can be installed in a core or linker to increase therigidity of the structure. Unsaturated bonds, such as alkenes andalkynes, may also or alternatively be installed. Such modifications mayensure the NHE-inhibiting compound is accessible for productive bindingwith its target. Furthermore, the hydrophilicity of the linkers may beimproved by adding hydrogen bond donor or acceptor motifs, or ionicmotifs such as amines that are protonated in the GI, or acids that aredeprotonated. Such modifications will increase the hydrophilicity of thecore or linker and help prevent hydrophobic collapse. Furthermore, suchmodifications will also contribute to the impermeability of theresulting compounds by increasing tPSA.

Specific examples of NHE-inhibiting small molecules modified consistentwith the principles detailed above are illustrated below. These moietiesdisplay functional groups that facilitate their appendage to “Z” (e.g.,a core group, Core, or linking group, L). These functional groups caninclude electrophiles, which can react with nucleophilic cores orlinkers, and nucleophiles, which can react with electrophilic cores orlinkers. Small molecule NHE inhibitors may be similarly derivatizedwith, for example, boronic acid groups which can then react withappropriate cores or linkers via palladium mediated cross-couplingreactions. The NHE inhibitor may also contain olefins which can thenreact with appropriate cores or linkers via olefin metathesis chemistry,or alkynes or azides which can then react with appropriate cores orlinkers via [2+3] cycloaddition. One skilled in the art may consider avariety of functional groups that will allow the facile and specificattachment of an NHE inhibiting small molecule to a desired core orlinker. Exemplary functionalized derivatives of NHEs include but are notlimited to the following:

wherein the variables in the above-noted structures (e.g., R, etc.) areas defined in U.S. Pat. No. 6,399,824, the entire contents of which areincorporated herein by reference for all relevant and consistentpurposes.

wherein the variables in the above-noted structures (e.g., R₇₋₉, etc.)are as defined in U.S. Pat. No. 6,911,453, the entire contents of which(and in particular the text of columns 1-4 therein) are incorporatedherein by reference for all relevant and consistent purposes.

wherein the variables in the above-noted structures (e.g., R₇₋₉, etc.)are as defined in U.S. Patent Application No. 2005/0020612 and U.S. Pat.No. 6,911,453, the entire contents of which (and in particular the textof columns 1-4 therein) are incorporated herein by reference for allrelevant and consistent purposes.

It is to be noted that one skilled in the art can envision a number ofcore or linker moieties that may be functionalized with an appropriateelectrophile or nucleophile. Shown below are a series of such compoundsselected based on several design considerations, including solubility,steric effects, and their ability to confer, or be consistent with,favorable structure-activity relationships. In this regard it is to befurther noted, however, that the structures provided below, and above,are for illustration purposes only, and therefore should not be viewedin a limiting sense.

Exemplary electrophilic and nucleophilic linker moieties include, butare not limited to, the linker moieties illustrated in the Examples andthe following:

Nucleophilic Linkers (for Use with Electrophilic NHE-InhibitoryDerivatives)

Electrophilic Linkers (for Use with Nucleophilic NHE-InhibitoryDerivatives)

The linking moiety, L, in each of the described embodiments (includingembodiments in which a NHE-inhibiting small molecule is linked to a coresuch as an atom, another small molecule, a polymer moiety, an oligomermoiety, or a non-repeating moiety) can be a chemical linker, such as abond or other moiety, for example, comprising about 1 to about 200atoms, or about 1 to about 100 atoms, or about 1 to about 50 atoms, thatcan be hydrophilic and/or hydrophobic. In one embodiment, the linkingmoiety can be a polymer moiety grafted onto a polymer backbone, forexample, using living free radical polymerization approaches known inthe art. Preferred L structures or moieties may also be selected from,for example, oligoethylene glycol, oligopeptide, oligoethyleneimine,oligotetramethylene glycol and oligocaprolactone.

As noted, the core moiety can be an atom, a small molecule, an oligomer,a dendrimer or a polymer moiety, in each case having one or more sitesof attachment for L. For example, the core moiety can be a non-repeatingmoiety (considered as a whole including linking points to theinhibitors), selected for example from the group consisting of alkyl,phenyl, aryl, alkenyl, alkynyl, heterocyclic, amine, ether, sulfide,disulfide, hydrazine, and any of the foregoing substituted with oxygen,sulfur, sulfonyl, phosphonyl, hydroxyl, alkoxyl, amine, thiol, ether,carbonyl, carboxyl, ester, amide, alkyl, alkenyl, alkynyl, aryl,heterocyclic, and moieties comprising combinations thereof (in eachpermutation). A non-repeating moiety can include repeating units (e.g.,methylene) within portions or segments thereof (e.g., within an alkylsegment), without having discrete repeat units that constitute themoiety as a whole (e.g., in the sense of a polymer or oligomer).

Exemplary core moieties include but are not limited to the core moietiesillustrated in the Examples and ether moieties, ester moieties, sulfidemoieties, disulfide moieties, amine moieties, aryl moieties, alkoxylmoieties, etc., such as, for example, the following:

wherein the broken bonds (i.e., those having a wavy bond,

, through them) are points of connection to either an NHE inhibitor or alinker moiety displaying an NHE inhibitor, where said points ofconnection can be made using chemistries and functional groups known tothe art of medicinal chemistry, and further wherein each p, q, r and sis an independently selected integer ranging from about 0 to about 48,preferably from about 0 to about 36, or from about 0 to about 24, orfrom about 0 to about 16. In some instances, each p, q, r and s can bean independently selected integer ranging from about 0 to 12.Additionally, R can be a substituent moiety generally selected fromhalide, hydroxyl, amine, thiol, ether, carbonyl, carboxyl, ester, amide,carbocyclic, heterocyclic, and moieties comprising combinations thereof.

In another approach, the core moiety is a dendrimer, defined as arepeatedly branched molecule (see, e.g., J. M. J. Fréchet, D. A.Tomalia, Dendrimers and Other Dendritic Polymers, John Wiley & Sons,Ltd. NY, N.Y., 2001) and schematically represented below:

In this approach, the NHE inhibiting small molecule is attached throughL to one, several or optionally all termini located at the periphery ofthe dendrimer. In another approach, a dendrimer building block nameddendron, and illustrated above, is used as a core, wherein the NHEinhibitor group is attached to one, several or optionally all terminilocated at the periphery of the dendron. The number of generationsherein is typically between about 0 and about 6, and preferably betweenabout 0 and about 3. (Generation is defined in, for example, J. M. J.Fréchet, D. A. Tomalia, Dendrimers and Other Dendritic Polimers, JohnWiley & Sons, Ltd. NY, N.Y.) Dendrimer and/or dendron structures arewell known in the art and include, for example, those shown in orillustrated by: (i) J. M. J. Fréchet, D. A. Tomalia, Dendrimers andOther Dendritic Polymers, John Wiley & Sons, Ltd. NY, N.Y.; (ii) GeorgeR Newkome, Charles N. Moorefield and Fritz Vogtle, Dendrimers andDendrons: Concepts. Syntheses, Applications, VCH VerlagsgesellschaftMbh; and, (iii) Boas, U., Christensen, J. B., Heegaard, P. M. H.,Dendrimers in Medicine and Biotechnology: New Molecular Tools, Springer,2006.

In yet another approach, the core moiety may be a polymer moiety or anoligomer moiety. The polymer or oligomer may, in each case, beindependently considered and comprise repeat units consisting of arepeat moiety selected from alkyl (e.g., —CH₂—), substituted alkyl(e.g., —CHR—, wherein, for example, R is hydroxy), alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, phenyl, aryl, heterocyclic,amine, ether, sulfide, disulfide, hydrazine, and any of the foregoingsubstituted with oxygen, sulfur, sulfonyl, phosphonyl, hydroxyl,alkoxyl, amine, thiol, ether, carbonyl, carboxyl, ester, amide, alkyl,alkenyl, alkynyl, aryl, heterocyclic, as well as moieties comprisingcombinations thereof. In still another approach, the core moietycomprises repeat units resulting from the polymerization of ethylenicmonomers (e.g., such as those ethylenic monomers listed elsewhere hereinbelow).

Preferred polymers for polymeric moieties useful in constructingsubstantially impermeable or substantially systemically non-bioavailableNHE-inhibiting compounds that are multivalent, for use in the treatmentvarious treatment methods disclosed herein, can be prepared by anysuitable technique, such as by free radical polymerization, condensationpolymerization, addition polymerization, ring-opening polymerization,and/or can be derived from naturally occurring polymers, such assaccharide polymers. Further, in some embodiments, any of these polymermoieties may be functionalized.

Examples of polysaccharides useful in preparation of such compoundsinclude but are not limited to materials from vegetable or animalorigin, including cellulose materials, hemicellulose, alkyl cellulose,hydroxyalkyl cellulose, carboxymethylcellulose, sulfoethylcellulose,starch, xylan, amylopectine, chondroitin, hyarulonate, heparin, guar,xanthan, mannan, galactomannan, chitin, and/or chitosan. More preferred,in at least some instances, are polymer moieties that do not degrade, orthat do not degrade significantly, under the physiological conditions ofthe GI tract (such as, for example, carboxymethylcellulose, chitosan,and sulfoethylcellulose).

When free radical polymerization is used, the polymer moiety can beprepared from various classes of monomers including, for example,acrylic, methacrylic, styrenic, vinylic, and dienic, whose typicalexamples are given thereafter: styrene, substituted styrene, alkylacrylate, substituted alkyl acrylate, alkyl methacrylate, substitutedalkyl methacrylate, acrylonitrile, methacrylonitrile, acrylamide,methacrylamide, N-alkylacrylamide, N-alkylmethacrylamide,N,N-dialkylacrylamide, N,N-dialkylmethacrylamide, isoprene, butadiene,ethylene, vinyl acetate, and combinations thereof. Functionalizedversions of these monomers may also be used and any of these monomersmay be used with other monomers as comonomers. For example, specificmonomers or comonomers that may be used in this disclosure includemethyl methacrylate, ethyl methacrylate, propyl methacrylate (allisomers), butyl methacrylate (all isomers), 2-ethylhexyl methacrylate,isobornyl methacrylate, methacrylic acid, benzyl methacrylate, phenylmethacrylate, methacrylonitrile, α-methylstyrene, methyl acrylate, ethylacrylate, propyl acrylate (all isomers), butyl acrylate (all isomers),2-ethylhexyl acrylate, isobornyl acrylate, acrylic acid, benzylacrylate, phenyl acrylate, acrylonitrile, styrene, glycidylmethacrylate, 2-hydroxyethyl methacrylate, hydroxypropyl methacrylate(all isomers), hydroxybutyl methacrylate (all isomers),N,N-dimethylaminoethyl methacrylate, N,N-diethylaminoethyl methacrylate,triethyleneglycol methacrylate, itaconic anhydride, itaconic acid,glycidyl acrylate, 2-hydroxyethyl acrylate, hydroxypropyl acrylate (allisomers), hydroxybutyl acrylate (all isomers), N,N-dimethylaminoethylacrylate, N,N-diethylaminoethyl acrylate, triethyleneglycol acrylate,methacrylamide, N-methylacrylamide, N,N-dimethylacrylamide,N-tert-butylmethacrylamide, N-n-butylmethacrylamide.N-methylolmethacrylamide, N-ethylolmethacrylamide,N-tert-butylacrylamide, N—N-butylacrylamide, N-methylolacrylamide,N-ethylolacrylamide, 4-acryloylmorpholine, vinyl benzoic acid (allisomers), diethylaminostyrene (all isomers), a-methylvinyl benzoic acid(all isomers), diethylamino α-methylstyrene (all isomers),p-vinylbenzene sulfonic acid, p-vinylbenzene sulfonic sodium salt,alkoxy and alkyl silane functional monomers, maleic anhydride,N-phenylmaleimide, N-butylmaleimide, butadiene, isoprene, chloroprene,ethylene, vinyl acetate, vinylformamide, allylamine, vinylpyridines (allisomers), fluorinated acrylate, methacrylates, and combinations thereof.Main chain heteroatom polymer moieties can also be used, includingpolyethyleneimine and polyethers such as polyethylene oxide andpolypropylene oxide, as well as copolymers thereof.

In one particular embodiment, the polymer to which the NHE inhibitorsmall molecule, NHE, is attached or otherwise a part of is a polyol(e.g., a polymer having a repeat unit of, for example, ahydroxyl-substituted alkyl, such as —CH(OH)—). Polyols, such as mono-and disaccharides, with or without reducing or reducible end groupsthereon, may be good candidates, for example, for installing additionalfunctionality that could render the compound substantially impermeable.

In one particular embodiment, the NHE inhibiting small molecule, NHE, isattached at one or both ends of the polymer chain. More specifically, inyet another alternative approach to the polyvalent embodiment of thepresent disclosure, a macromolecule (e.g., a polymer or oligomer) havingone of the following exemplary structures may be designed andconstructed as described herein:

It is to be further noted that the repeat moiety in Formulas (XIIA) or(XIIB) generally encompasses repeating units of polymers and copolymersproduced by methods referred to herein above.

It is to be noted that the various properties of the oligomers andpolymers that form the core moiety as disclosed herein above may beoptim/zed for a given use or application using experimental means andprinciples generally known in the art. For example, the overallmolecular weight of the compounds or structures presented herein abovemay be selected so as to achieve non-absorbability, inhibitionpersistence and/or potency.

Additionally, with respect to those polymeric embodiments that encompassor include the compounds generally represented by the structure ofFormula (I) herein, and/or those disclosed for example in the manypatents and patent applications cited herein (see, e.g., U.S. Pat. Nos.5,866,610; 6,399,824; 6,911,453; 6,703,405; 6,005,010; 6,887,870;6,737,423; 7,326,705; 55,824,691 (WO94/026709); U.S. Pat. No. 6,399,824(WO02/024637); US 2004/0339001 (WO02/020496); US 2005/0020612(WO03/055490); WO01/072742; CA 2387529 (WO01021582): CA 02241531(WO97/024113); US 2005/0113396 (WO3/051866); US2005/0020612;US2005/0054705; US2008/0194621; US2007/0225323; US2004/0039001;US2004/0224965; US2005/0113396; US2007/0135383; US2007/0135385;US2005/0244367; US2007/0270414; and CA 2177007 (EP0744397), the entirecontents of which are incorporated herein by reference for all relevantand consistent purposes), such as those wherein these compounds orstructures are pendants off of a polymeric backbone or chain, thecomposition of the polymeric backbone or chain, as well as the overallsize or molecular weight of the polymer, and/or the number of pendantmolecules present thereon, may be selected according to variousprinciples known in the art in view of the intended application or use.

With respect to the polymer composition of the NHE inhibiting compound,it is to be noted that a number of polymers can be used including, forexample, synthetic and/or naturally occurring aliphatic, alicyclic,and/or aromatic polymers. In preferred embodiments, the polymer moietyis stable under physiological conditions of the GI tract. By “stable” itis meant that the polymer moiety does not degrade or does not degradesignificantly or essentially does not degrade under the physiologicalconditions of the GI tract. For instance, at least about 90%, preferablyat least about 95%, and more preferably at least about 98%, and evenmore preferably at least about 99% of the polymer moiety remainsun-degraded or intact after at least about 5 hours, at least about 12hours, at least about 18 hours, at least about 24 hours, or at leastabout 48 hours of residence in a gastrointestinal tract. Stability in agastrointestinal tract can be evaluated using gastrointestinal mimics,e.g., gastric mimics or intestinal mimics of the small intestine, whichapproximately model the physiological conditions at one or morelocations therein.

Polymer moieties detailed herein for use as the core moiety can behydrophobic, hydrophilic, amphiphilic, uncharged or non-ionic,negatively or positively charged, or a combination thereof.Additionally, the polymer architecture of the polymer moiety can belinear, grafted, comb, block, star and/or dendritic, preferably selectedto produce desired solubility and/or stability characteristics asdescribed above.

Additionally or alternatively, modifications may be made toNHE-inhibiting small molecules that increase tPSA, thus contributing tothe impermeability of the resulting compounds. Such modificationspreferably include addition of di-anions, such as phosphonates,malonates, sulfonates and the like, and polyols such as carbohydratesand the like. Exemplary derivatives of NHEs with increased tPSA includebut are not limited to the following:

B. Preferred Embodiments

In one or more particularly preferred embodiments of the presentdisclosure, the “NHE-Z” molecule is polyvalent; that is, the moleculecontains two or more moieties that effectively acts to inhibitNHE-mediated antiport of sodium ions and hydrogen ions. In suchembodiments, the NHE-Z molecule may be selected, for example, from oneof the following Formulas (IV), (V), (VI) or (VII):

wherein: each R₁, R₂, R₃, R₅ and R₉ are independently selected from H,halogen, —NR₇(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇,—SR₇, —O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or L, provided at least one is L, whereinL is selected from the group consisting of substituted or unsubstitutedhydrocarbyl, heterohydrocarbyl, polyalkylene glycol and polyols, andfurther wherein L links the repeat unit to at least one other repeatunit and/or at least one other Core moiety independently selected fromsubstituted or unsubstituted hydrocarbyl, heterohydrocarbyl,polyalkylene glycol, polyols, polyamines, or polyacrylamides, of thepolyvalent compound; R₄ is selected from H, C₁-C₇ alkyl or L, where L isas described above; R₆ is absent or selected from H and C₁-C₇ alkyl;and, Ar1 and Ar2 independently represent an aromatic ring, oralternatively a heteroaromatic ring wherein one or more of the carbonatoms therein is replaced with a N, O or S atom;

wherein: each R₁, R₂, R₃, and R₅ are optionally linked to the ring Ar1by a heterocyclic linker, and further are independently selected from H,—NR₇(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇, —SR₇,—O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or L, provided at least one is L, whereinL is selected from the group consisting of substituted or unsubstitutedhydrocarbyl, heterohydrocarbyl, polyalkylene glycol and polyols, andfurther wherein L links the repeat unit to at least one other repeatunit and/or at least one other Core moiety independently selected fromsubstituted or unsubstituted hydrocarbyl, heterohydrocarbyl,polyalkylene glycol, polyols, polyamines, or polyacrylamides, of thepolyvalent compound; R₄ and R₁₂ are independently selected from H or L,where L is as defined above; R₁₀ and R₁₁, when presented, areindependently selected from H and C₁-C₇ alkyl; and, Ar1 and Ar2independently represent an aromatic ring, or alternatively aheteroaromatic ring wherein one or more of the carbon atoms therein isreplaced with a N, O or S atom;

wherein: each X is a halogen atom, which may be the same or different;R₁ is selected from —SO₂—NR₇R₈, —NR₇(CO)R₈, —(CO)NR₇R₈, —NR₇SO₂R₈,—NR₇R₈, —OR₇—, —SR₇—, —O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇and R₈ are independently selected from H or L, provided at least one isL, wherein L is selected from the group consisting of substituted orunsubstituted hydrocarbyl, heterohydrocarbyl, polyalkylene glycol andpolyols, and further wherein L links the repeat unit to at least oneother repeat unit and/or at least one other Core moiety independentlyselected from substituted or unsubstituted hydrocarbyl,heterohydrocarbyl, polyalkylene glycol, polyols, polyamines, orpolyacrylamides, of the polyvalent compound; R₃ is selected from H or L,where L is as described above; R₁₃ is selected from substituted orunsubstituted C₁-C₈ alkyl; R₂ and R₁₂ are independently selected from Hor L, wherein L is as described above; R₁₀ and R₁₁, when present, areindependently selected from H and C₁-C₇ alkyl; Ar1 represents anaromatic ring, or alternatively a heteroaromatic ring wherein one ormore of the carbon atoms therein is replaced with a N. O or S atom; andAr2 represents an aromatic ring, or alternatively a heteroaromatic ringwherein one or more of the carbon atoms therein is replaced with a N, Oor S atom.

In one particular embodiment for the structure of Formula (V), one ofR₁, R₂ and R₃ is linked to the ring Ar1, and/or R₅ is linked to the ringAr2, by a heterocyclic linker having the structure:

wherein R represents R₁, R₂, R₃, or R₅ bound thereto.

In one particular embodiment, the NHE-inhibiting small molecule has thestructure of Formula (IV):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein: each R₁, R₂, R₃, R₅ and R₉ are independently selected from H,halogen, —NR₇(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇,—SR₇, —O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or a bond linking the NHE-inhibiting smallmolecule to L, provided at least one is a bond linking theNHE-inhibiting small molecule to L; R₄ is selected from H, C₁-C₇ alkyl,or a bond linking the NHE-inhibiting small molecule to L; R₆ is absentor selected from H and C₁-C₇ alkyl; and Ar1 and Ar2 independentlyrepresent an aromatic ring or a heteroaromatic ring.

In further particular embodiments of the above embodiment, theNHE-inhibiting small molecule has the following structure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein: each R₁, R₂ and R₃ are independently selected from H, halogen,—NR₇(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇, —SR₇,—O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or a bond linking the NHE-inhibiting smallmolecule to L, provided at least one is a bond linking theNHE-inhibiting small molecule to L.

In further particular embodiments of the above embodiment, theNHE-inhibiting small molecule has one of the following structures:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.

In further particular embodiments of the above embodiment, L is apolyalkylene glycol linker, such as a polyethylene glycol linker.

In further particular embodiments of the above embodiment, n is 2.

In further particular embodiments of the above embodiment, the Core hasthe following structure:

wherein: X is selected from the group consisting of a bond. —O—, —NH—,—S—, C₁₋₆alkylene, —NHC(═O)—, —C(═O)NH—, —NHC(═O)NH—, —SO₂NH—, and—NHSO₂—; Y is selected from the group consisting of a bond, optionallysubstituted C₁₋₈alkylene, optionally substituted aryl, optionallysubstituted heteroaryl, a polyethylene glycol linker,—(CH₂)₁₋₆O(CH₂)₁₋₆— and —(CH₂)₁₋₆NY₁(CH₂)₁₋₆—; and Y₁ is selected fromthe group consisting of hydrogen, optionally substituted C₁₋₈alkyl,optionally substituted aryl or optionally substituted heteroaryl.

In further particular embodiments of the above embodiment, the Core isselected from the group consisting of:

III. Terminology, Physical and Performance Properties A. Terminology

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open,inclusive sense, that is as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

“Amino” refers to the —NH₂ radical.

“Cyano” refers to the —CN radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Imino” refers to the ═NH substituent.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Thioxo” refers to the ═S substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds),having from one to twelve carbon atoms (C₁-C₁₂ alkyl), preferably one toeight carbon atoms (C₁-C₈ alkyl) or one to six carbon atoms (C₁-C₆alkyl), and which is attached to the rest of the molecule by a singlebond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl,2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl,penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and thelike. Unless stated otherwise specifically in the specification, analkyl group may be optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double and/or triple bonds), andhaving from one to twelve carbon atoms, e.g., methylene, ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. The alkylene chain is attachedto the rest of the molecule through a single or double bond and to theradical group through a single or double bond. The points of attachmentof the alkylene chain to the rest of the molecule and to the radicalgroup can be through one carbon or any two carbons within the chain.Unless stated otherwise specifically in the specification, an alkylenechain may be optionally substituted.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, an alkoxygroup may be optionally substituted.

“Alkylamino” refers to a radical of the formula —NHR_(a) or —NR_(a)R_(a)where each R_(a) is, independently, an alkyl radical as defined abovecontaining one to twelve carbon atoms. Unless stated otherwisespecifically in the specification, an alkylamino group may be optionallysubstituted.

“Thioalkyl” refers to a radical of the formula —SR_(a) where R_(a) is analkyl radical as defined above containing one to twelve carbon atoms.Unless stated otherwise specifically in the specification, a thioalkylgroup may be optionally substituted.

“Aryl” refers to a hydrocarbon ring system radical comprising hydrogen,6 to 18 carbon atoms and at least one aromatic ring. For purposes ofthis invention, the aryl radical may be a monocyclic, bicyclic,tricyclic or tetracyclic ring system, which may include fused or bridgedring systems. Aryl radicals include, but are not limited to, arylradicals derived from aceanthrylene, acenaphthylene, acephenanthrylene,anthracene, azulene, benzene, chrysene, fluoranthene, fluorene,as-indacene, s-indacene, indane, indene, naphthalene, phenalene,phenanthrene, pleiadene, pyrene, and triphenylene. Unless statedotherwise specifically in the specification, the term “aryl” or theprefix “ar-” (such as in “aralkyl”) is meant to include aryl radicalsthat are optionally substituted.

“Aralkyl” refers to a radical of the formula —R_(b)—R_(c) where R_(b) isan alkylene chain as defined above and R_(c) is one or more arylradicals as defined above, for example, benzyl, diphenylmethyl and thelike. Unless stated otherwise specifically in the specification, anaralkyl group may be optionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon radical consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic radicalsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example,adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl,and the like. Unless otherwise stated specifically in the specification,a cycloalkyl group may be optionally substituted.

“Cycloalkylalkyl” refers to a radical of the formula —R_(b)R_(d) whereR_(d) is an alkylene chain as defined above and R_(g) is a cycloalkylradical as defined above. Unless stated otherwise specifically in thespecification, a cycloalkylalkyl group may be optionally substituted.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure in the compounds of the invention. When thefused ring is a heterocyclyl ring or a heteroaryl ring, any carbon atomon the existing ring structure which becomes part of the fusedheterocyclyl ring or the fused heteroaryl ring may be replaced with anitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl,1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and thelike. Unless stated otherwise specifically in the specification, ahaloalkyl group may be optionally substituted.

“Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to18-membered non-aromatic ring radical which consists of two to twelvecarbon atoms and from one to six heteroatoms selected from the groupconsisting of nitrogen, oxygen and sulfur. Unless stated otherwisespecifically in the specification, the heterocyclyl radical may be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which mayinclude fused or bridged ring systems; and the nitrogen, carbon orsulfur atoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the heterocyclylradical may be partially or fully saturated. Examples of suchheterocyclyl radicals include, but are not limited to, dioxolanyl,thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl,imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl,octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in thespecification, Unless stated otherwise specifically in thespecification, a heterocyclyl group may be optionally substituted.

“N-heterocyclyl” refers to a heterocyclyl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heterocyclyl radical to the rest of the molecule is through anitrogen atom in the heterocyclyl radical. Unless stated otherwisespecifically in the specification, a N-heterocyclyl group may beoptionally substituted.

“Heterocyclylalkyl” refers to a radical of the formula —R_(b)R_(e) whereR_(b) is an alkylene chain as defined above and R_(e) is a heterocyclylradical as defined above, and if the heterocyclyl is anitrogen-containing heterocyclyl, the heterocyclyl may be attached tothe alkyl radical at the nitrogen atom. Unless stated otherwisespecifically in the specification, a heterocyclylalkyl group may beoptionally substituted.

“Heteroaryl” refers to a 5- to 14-membered ring system radicalcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, and at least one aromatic ring. For purposes of this invention,the heteroaryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwisespecifically in the specification, a heteroaryl group may be optionallysubstituted.

“N-heteroaryl” refers to a heteroaryl radical as defined abovecontaining at least one nitrogen and where the point of attachment ofthe heteroaryl radical to the rest of the molecule is through a nitrogenatom in the heteroaryl radical. Unless stated otherwise specifically inthe specification, an N-heteroaryl group may be optionally substituted.

“Heteroarylalkl” refers to a radical of the formula —R_(b)R_(f) whereR_(b) is an alkylene chain as defined above and R_(f) is a heteroarylradical as defined above. Unless stated otherwise specifically in thespecification, a heteroarylalkyl group may be optionally substituted.

The term “substituted” used herein means any of the above groups (i.e.,alkyl, alkylene, alkoxy, alkylamino, thioalkyl, aryl, aralkyl,cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl)wherein at least one hydrogen atom is replaced by a bond to anon-hydrogen atoms such as, but not limited to: a halogen atom such asF, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups,alkoxy groups, and ester groups; a sulfur atom in groups such as thiolgroups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxidegroups; a nitrogen atom in groups such as amines, amides, alkylamines,dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides,imides, and enamines; a silicon atom in groups such as trialkylsilylgroups, dialkylarylsilyl groups, alkyldiarylsilyl groups, andtriarylsilyl groups; and other heteroatoms in various other groups.“Substituted” also means any of the above groups in which one or morehydrogen atoms are replaced by a higher-order bond (e.g., a double- ortriple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl,and ester groups; and nitrogen in groups such as imines, oximes,hydrazones, and nitriles. For example, “substituted” includes any of theabove groups in which one or more hydrogen atoms are replaced with—NR_(g)R_(h), —NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g),—SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and—SO₂NR_(g)R_(h). “Substituted” also means any of the above groups inwhich one or more hydrogen atoms are replaced with —C(═O)R_(g),—C(═O)OR_(g). —C(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h),—(CH₂CH₂O)₂₋₁₀R_(g). In the foregoing, R_(g) and R_(h) are the same ordifferent and independently hydrogen, alkyl, alkoxy, alkylamino,thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl,heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl,N-heteroaryl and/or heteroarylalkyl. “Substituted” further means any ofthe above groups in which one or more hydrogen atoms are replaced by abond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo,alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl,cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl,heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkylgroup. In addition, each of the foregoing substituents may also beoptionally substituted with one or more of the above substituents.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the invention. Prodrugs are typically rapidly transformed invivo to yield the parent compound of the invention, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

The term “prodrug” is aso meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of the invention. Prodrugs include compounds of theinvention wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the compound of the invention isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol or amide derivatives of amine functional groupsin the compounds of the invention and the like.

The invention disclosed herein is also meant to encompass the in vivometabolic products of the disclosed compounds. Such products may resultfrom, for example, the oxidation, reduction, hydrolysis, amidation,esterification, and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes compoundsproduced by a process comprising administering a compound of thisinvention to a mammal for a period of time sufficient to yield ametabolic product thereof. Such products are typically identified byadministering a radiolabelled compound of the invention in a detectabledose to an animal, such as rat, mouse, guinea pig, monkey, or to human,allowing sufficient time for metabolism to occur, and isolating itsconversion products from the urine, blood or other biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Optional” or “optionally” means that the subsequently described eventor circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, “optionally substituted aryl” means that thearyl radical may or may not be substituted and that the descriptionincludes both substituted aryl radicals and aryl radicals having nosubstitution.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of theinvention. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of the invention may be truesolvates, while in other cases, the compound of the invention may merelyretain adventitious water or be a mixture of water plus someadventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

The compounds of the invention, or their pharmaceutically acceptablesalts may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centres of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds.

In accordance with the present disclosure, the compounds describedherein are designed to be substantially active or localized in thegastrointestinal lumen of a human or animal subject. The term“gastrointestinal lumen” is used interchangeably herein with the term“lumen.” to refer to the space or cavity within a gastrointestinal tract(GI tract, which can also be referred to as the gut), delimited by theapical membrane of GI epithelial cells of the subject. In someembodiments, the compounds are not absorbed through the layer ofepithelial cells of the GI tract (also known as the GI epithelium).“Gastrointestinal mucosa” refers to the layer(s) of cells separating thegastrointestinal lumen from the rest of the body and includes gastricand intestinal mucosa, such as the mucosa of the small intestine. A“gastrointestinal epithelial cell” or a “gut epithelial cell” as usedherein refers to any epithelial cell on the surface of thegastrointestinal mucosa that faces the lumen of the gastrointestinaltract, including, for example, an epithelial cell of the stomach, anintestinal epithelial cell, a colonic epithelial cell, and the like.

“Substantially systemically non-bioavailable” and/or “substantiallyimpermeable” as used herein (as well as variations thereof) generallyrefer to situations in which a statistically significant amount, and insome embodiments essentially all of the compound of the presentdisclosure (which includes the NHE-inhibitor small molecule), remains inthe gastrointestinal lumen. For example, in accordance with one or moreembodiments of the present disclosure, preferably at least about 70%,about 80%, about 90%, about 95%, about 98%, about 99%, or even about99.5%, of the compound remains in the gastrointestinal lumen. In suchcases, localization to the gastrointestinal lumen refers to reducing netmovement across a gastrointestinal layer of epithelial cells, forexample, by way of both transcellular and paracellular transport, aswell as by active and/or passive transport. The compound in suchembodiments is hindered from net permeation of a layer ofgastrointestinal epithelial cells in transcellular transport, forexample, through an apical membrane of an epithelial cell of the smallintestine. The compound in these embodiments is also hindered from netpermeation through the “tight junctions” in paracellular transportbetween gastrointestinal epithelial cells lining the lumen.

In this regard it is to be noted that, in one particular embodiment, thecompound is essentially not absorbed at all by the GI tract orgastrointestinal lumen. As used herein, the terms “substantiallyimpermeable” or “substantially systemically non-bioavailable” refers toembodiments wherein no detectable amount of absorption or permeation orsystemic exposure of the compound is detected, using means generallyknown in the art.

In this regard it is to be further noted, however, that in alternativeembodiments “substantially impermeable” or “substantially systemicallynon-bioavailable” provides or allows for some limited absorption in theGI tract, and more particularly the gut epithelium, to occur (e.g., somedetectable amount of absorption, such as for example at least about0.1%, 0.5%, 1% or more and less than about 30%, 20%, 10%, 5%, etc., therange of absorption being for example between about 1% and 30%, or 5%and 20%, etc.; stated another way, “substantially impermeable” or“substantially systemically non-bioavailable” refers to compounds thatexhibit some detectable permeability to an epithelium layer of cells inthe GI tract of less than about 20% of the administered compound (e.g.,less than about 15%, about 10%, or even about 5%, and for examplegreater than about 0.5%, or 1%), but then are cleared by the liver(i.e., hepatic extraction) and/or the kidney (i.e., renal excretion).

B. Permeability

In this regard it is to be noted that, in various embodiments, theability of the compound to be substantially systemicallynon-bioavailable is based on the compound charge, size, and/or otherphysicochemical parameters (e.g., polar surface area, number of hydrogenbond donors and/or acceptors therein, number of freely rotatable bonds,etc.). More specifically, it is to be noted that the absorptioncharacter of a compound can be selected by applying principles ofpharmacodynamics, for example, by applying Lipinski's rule, also knownas “the rule of five.” Although not a rule, but rather a set ofguidelines, Lipinski shows that small molecule drugs with (i) amolecular weight, (ii) a number of hydrogen bond donors, (iii) a numberof hydrogen bond acceptors, and/or (iv) a water/octanol partitioncoefficient (Moriguchi Log P), greater than a certain threshold value,generally do not show significant systemic concentration (i.e., aregenerally not absorbed to any significant degree). (See, e.g., Lipinskiet al., Advanced Drug Delivery Reviews, 46, 2001 3-26, incorporatedherein by reference.) Accordingly, substantially systemicallynon-bioavailable compounds (e.g., substantially systemicallynon-bioavailable NHE inhibitor compounds) can be designed to havemolecular structures exceeding one or more of Lipinski's thresholdvalues. (See also Lipinski et al., Experimental and ComputationalApproaches to Estimate Solubility and Permeability in Drug Discovery andDevelopment Settings, Adv. Drug Delivery Reviews, 46:3-26 (2001): andLipinski, Drug-like Properties and the Causes of Poor Solubility andPoor Permeability. J. Pharm. & Toxicol. Methods, 44:235-249 (2000),incorporated herein by reference.) In some embodiments, for example, asubstantially impermeable or substantially systemically non-bioavailableNHE inhibitor compound of the present disclosure can be constructed tofeature one or more of the following characteristics: (i) a MW greaterthan about 500 Da, about 1000 Da, about 2500 Da, about 5000 Da, about10,000 Da or more (in the non-salt form of the compound); (ii) a totalnumber of NH and/or OH and/or other potential hydrogen bond donorsgreater than about 5, about 10, about 15 or more; (iii) a total numberof O atoms and/or N atoms and/or other potential hydrogen bond acceptorsgreater than about 5, about 10, about 15 or more; and/or (iv) aMoriguchi partition coefficient greater than about 10⁵ (i.e., Log Pgreater than about 5, about 6, about 7, etc.), or alternatively lessthan about 10 (i.e., a Log P of less than 1, or even 0).

In view of the foregoing, and as previously noted herein, essentiallyany known NHE inhibitor small molecule (described herein and/or in theart) can be used in designing a substantially systemicallynon-bioavailable NHE inhibitor molecular structure, in accordance withthe present disclosure. In addition to the parameters noted above, themolecular polar surface area (i.e., “PSA”), which may be characterizedas the surface belonging to polar atoms, is a descriptor that has alsobeen shown to correlate well with passive transport through membranesand, therefore, allows prediction of transport properties of drugs. Ithas been successfully applied for the prediction of intestinalabsorption and Caco2 cell monolayer penetration. (For Caco2 cellmonolayer penetration test details, see for example the description ofthe Caco2 Model provided in Example 31 of U.S. Pat. No. 6,737,423, theentire contents of which are incorporated herein by reference for allrelevant and consistent purposes, and the text of Example 31 inparticular, which may be applied for example to the evaluation ortesting of the compounds of the present disclosure.) PSA is expressed inÅ² (squared angstroms) and is computed from a three-dimensionalmolecular representation. A fast calculation method is now available(see, e.g., Ertl et al., Journal of Medicinal Chemistry, 2000, 43,3714-3717, the entire contents of which are incorporated herein byreference for all relevant and consistent purposes) using a desktopcomputer and commercially available chemical graphic tools packages,such as ChemDraw. The term “topological PSA” (tPSA) has been coined forthis fast-calculation method, tPSA is well correlated with humanabsorption data with common drugs (see, e.g., Table 2, below):

TABLE 2 name % FA^(a) TPSA^(b) metoprolol 102 50.7 nordiazepam 99 41.5diazepam 97 32.7 oxprenolol 97 50.7 phenazone 97 26.9 oxazepam 97 61.7alprenolol 96 41.9 practolol 95 70.6 pindolol 92 57.3 ciprofloxacin 6974.6 metolazone 64 92.5 tranexamic acid 55 63.3 atenolol 54 84.6sulpiride 36 101.7 mannitol 26 121.4 foscarnet 17 94.8 sulfasalazine 12141.3 olsalazine 2.3 139.8 lactulose 0.6 197.4 raffinose 0.3 268.7(from Ertl et al., J. Med Chem., 2000, 43:3714-3717). Accordingly, insome preferred embodiments, the compounds of the present disclosure maybe constructed to exhibit a tPSA value greater than about 100 Å², about120 Å², about 130 Å², or about 140 Å², and in some instances about 150Å², about 200 Å², about 250 Å², about 270 Å², about 300 Å², about 400Å², or even about 500 Å², such that the compounds are substantiallyimpermeable or substantially systemically non-bioavailable (as definedelsewhere herein).

Because there are exceptions to Lipinski's “rule,” or the tPSA model,the permeability properties of the compounds of the present disclosuremay be screened experimentally. The permeability coefficient can bedetermined by methods known to those of skill in the art, including forexample by Caco-2 cell permeability assay and/or using an artificialmembrane as a model of a gastrointestinal epithelial cell. (Aspreviously noted above, see for example U.S. Pat. No. 6,737,423, Example31 for a description of the Caco-2 Model, which is incorporated hereinby reference). A synthetic membrane impregnated with, for example,lecithin and/or dodecane to mimic the net permeability characteristicsof a gastrointestinal mucosa, may be utilized as a model of agastrointestinal mucosa. The membrane can be used to separate acompartment containing the compound of the present disclosure from acompartment where the rate of permeation will be monitored. Also,parallel artificial membrane permeability assays (PAMPA) can beperformed. Such in vitro measurements can reasonably indicate actualpermeability in vivo. (See, for example, Wohnsland et al., J Med. Chem.,2001, 44:923-930; Schmidt et al., Millipore Corp. Application Note,2002, no AN1725EN00, and no ANI728EN00, incorporated herein byreference.)

Accordingly, in some embodiments, the compounds utilized in the methodsof the present disclosure may have a permeability coefficient, P_(app),of less than about 100×10⁻⁶ cm/s, or less than about 10×10⁻⁶ cm/s, orless than about 1×10⁻⁶ cm/s, or less than about 0.1×10⁻⁶ cm/s, whenmeasured using means known in the art (such as for example thepermeability experiment described in Wohnsland et al., J. Med. Chem.,2001, 44, 923-930, the contents of which is incorporated herein byreference).

As previously noted, in accordance with the present disclosure. NHEinhibitor small molecules are modified as described above to hinder thenet absorption through a layer of gut epithelial cells, rendering themsubstantially systemically non-bioavailable. In some particularembodiments, the compounds of the present disclosure comprise anNHE-inhibiting small molecule linked, coupled or otherwise attached to amoiety Z, which may be an oligomer moiety, a polymer moiety, ahydrophobic moiety, a hydrophilic moiety, and/or a charged moiety, whichrenders the overall compound substantially impermeable or substantiallysystemically non-bioavailable. In some preferred embodiments, theNHE-inhibiting small molecule is coupled to a multimer or polymerportion or moiety, such that the resulting NHE-Z molecule issubstantially impermeable or substantially systemicallynon-bioavailable. The multimer or polymer portion or moiety may be of amolecular weight greater than about 500 Daltons (Da), about 1000 Da,about 2500 Da, about 5000 Da, about 10,000 Da or more, and in particularmay have a molecular weight in the range of about 1000 Daltons (Da) toabout 500,000 Da, preferably in the range of about 5000 to about 200,000Da, and more preferably may have a molecular weight that is sufficientlyhigh to essentially preclude any net absorption through a layer of gutepithelial cells of the compound. For example, an NHE-inhibiting smallmolecule may be linked to at least one repeat unit of a polymer portionor moiety according, for example, to the structure of Formula (XIIA) orFormula (XIIB), as illustrated herein. In these or other particularembodiments, the NHE-inhibiting small molecule is modified as describedherein to substantially hinder its net absorption through a layer of gutepithelial cells and may comprise, for example, a NHE-inhibitingcompound linked, coupled or otherwise attached to a substantiallyimpermeable or substantially systemically non-bioavailable “Core”moiety, as described above.

C. Persistent Inhibitory Effect

In other embodiments, the substantially impermeable or substantiallysystemically non-bioavailable NHE-inhibiting compounds utilized in thetreatment methods of the present disclosure may additionally exhibit apersistent inhibitor effect. This effect manifests itself when theinhibitory action of a compound at a certain concentration inequilibrium with the epithelial cell (e.g., at or above its inhibitoryconcentration, IC) does not revert to baseline (i.e., sodium transportwithout inhibitor) after the compound is depleted by simple washing ofthe luminal content.

This effect can be interpreted as a result of the tight binding of theNHE-inhibiting compounds to the NHE protein at the intestinal apicalside of the gut epithelial cell. The binding can be considered asquasi-irreversible to the extent that, after the compound has beencontacted with the gut epithelial cell and subsequently washed off saidgut epithelial cell, the flux of sodium transport is still significantlylower than in the control without the compound. This persistentinhibitory effect has the clear advantage of maintaining drug activitywithin the GI tract even though the residence time of the active in theupper GI tract is short, and when no entero-biliary recycling process iseffective to replenish the compound concentration near its site ofaction.

Such a persistent inhibitory effect has an obvious advantage in terms ofpatient compliance, but also in limiting drug exposure within the GItract.

The persistence effect can be determined using in vitro methods; in oneinstance, cell lines expressing NHE transporters are split in differentvials and treated with a NHE-inhibiting compound and sodium solution tomeasure the rate of sodium uptake. The cells in one set of vials arewashed for different periods of time to remove the inhibitor, and sodiumuptake measurement is repeated after the washing. Compounds thatmaintain their inhibitory effect after multiple/lengthy washing steps(compared to the inhibitory effect measured in the vials where washingdoes not occur) are persistent inhibitors. Persistence effect can alsobe characterized ex vive by using the everted sac technique, wherebytransport of Na is monitored using an excised segment of GI perfusedwith a solution containing the inhibitor and shortly after flushing thebathing solution with a buffer solution free from inhibitor. Apersistence effect can also be characterized in vivo by observing thetime needed for sodium balance to return to normal when the inhibitortreatment is discontinued. The limit of the method resides in the factthat apical cells (and therefore apical NHE transporters) are sloughedoff after a period of 3 to 4 days, the typical turnover time of gutepithelial cells. A persistence effect can be achieved by increasing theresidence time of the active compound at the apical surface of the gutepithelial cells; this can be obtained by designing NHE antiportinhibitors with several NHE inhibiting moieties built-in the smallmolecule or oligomer (wherein “several” as used herein typically meansat least about 2, about 4, about 6 or more). Examples of such structuresin the context of analogs of the antibiotic vancomycin are given inGriffin, et al., J. Am. Chem. Soc., 2003, 125, 6517-6531. Alternativelythe compound comprises groups that contribute to increase the affinitytowards the gut epithelial cell so as to increase the time of contactwith the gut epithelial cell surface. Such groups are referred to asbeing “mucoadhesive.” More specifically, the Core or L moiety can besubstituted by such mucoadhesive groups, such as polyacrylates,partially deacetylated chitosan or polyalkylene glycol. (See also Patil,S. B. et al., Curr. Drug. Deliv., 2008, October 5(4), pp. 312-8.)

D. GI Enzyme Resistance

Because the compounds utilized in the treatment methods of the presentdisclosure are preferably substantially systemically non-bioavailable,and/or preferably exhibit a persistent inhibitory effect, it is alsodesirable that, during their prolonged residence time in the gut, thesecompounds sustain the hydrolytic conditions prevailing in the upper GItract. In such embodiments, compounds of the present disclosure areresistant to enzymatic metabolism. For example, administered compoundsare preferably resistant to the activity of P450 enzymes, glucurosyltransferases, sulfotransferases, glutathione S-transferases, and thelike, in the intestinal mucosa, as well as gastric (e.g., gastriclipase, and pepsine), pancreatic (e.g., trypsin, triglyceride pancreaticlipase, phospholipase A2, endonucleases, nucleotidases, andalpha-amylase), and brush-border enzymes (e.g., alkaline phosphatase,glycosidases, and proteases) generally known in the art.

The compounds that are utilized in methods of the present disclosure arealso preferably resistant to metabolism by the bacterial flora of thegut; that is, the compounds are not substrates for enzymes produced bybacterial flora. In addition, the compounds administered in accordancewith the methods of the present disclosure may be substantially inactivetowards the gastrointestinal flora, and do not disrupt bacterial growthor survival. As a result, in various embodiments herein, the minimalinhibitory concentration (or “MIC”) against GI flora is desirablygreater than about 15 μg/ml, about 30 μg/ml, about 60 μg/ml, about 120μg/ml, or even about 240 μg/ml, the MIC in various embodiments being forexample between about 16 and about 32 μg/ml, or between about 64 andabout 128 μg/ml, or greater than about 256 μg/ml.

To one skilled in the art of medicinal chemistry, metabolic stabilitycan be achieved in a number of ways. Functionality susceptible toP450-mediated oxidation can be protected by, for example, blocking thepoint of metabolism with a halogen or other functional group.Alternatively, electron withdrawing groups can be added to a conjugatedsystem to generally provide protection to oxidation by reducing theelectrophilicity of the compound. Proteolytic stability can be achievedby avoiding secondary amide bonds, or by incorporating changes instereochemistry or other modifications that prevent the drug fromotherwise being recognized as a substrate by the metabolizing enzyme.

E. Sodium and/or Fluid Output

It is also to be noted that, in various embodiments of the presentdisclosure, one or more of the NHE-Z inhibiting compounds (monovalent ordivalent) detailed herein, when administered either alone or incombination with one or more additional pharmaceutically activecompounds or agents (including, for example, a fluid-absorbing polymer)to a patient in need thereof, may act to increase the patient's dailyfecal output of sodium by at least about 20, about 30 mmol, about 40mmol, about 50 mmol, about 60 mmol, about 70 mmol, about 80 mmol, about90 mmol, about 100 mmol, about 125 mmol, about 150 mmol or more, theincrease being for example within the range of from about 20 to about150 mmol/day, or from about 25 to about 100 mmol/day, or from about 30to about 60 mmol/day

Additionally, or alternatively, it is also to be noted that, in variousembodiments of the present disclosure, one or more of the NHE-Zinhibiting compounds (monovalent or divalent) detailed herein, whenadministered either alone or in combination with one or more additionalpharmaceutically active compounds or agents (including, for example, afluid-absorbing polymer) to a patent in need thereof, may act toincrease the patient's daily fluid output by at least about 100 ml,about 200 ml, about 300 ml, about 400 ml, about 500 ml, about 600 ml,about 700 ml, about 800 ml, about 900 ml, about 1000 ml or more, theincrease being for example within the range of from about 100 to about1000 ml/day, or from about 150 to about 750 ml/day, or from about 200 toabout 500 ml/day (assuming isotonic fluid).

F. C_(max) and IC₅₀

It is also to be noted that, in various embodiments of the presentdisclosure, one or more of the NHE-Z inhibiting compounds (monovalent ordivalent) detailed herein, when administered either alone or incombination with one or more additional pharmaceutically activecompounds or agents (including, for example, a fluid-absorbing polymer)to a patient in need thereof at a dose resulting in at least a 10%increase in fecal water content, has a C_(max) that is less than theIC₅₀ for NHE-3, more specifically, less than about 10× (10 times) theIC₅₀, and, more specifically still, less than about 100× (100 times) theIC₅₀.

Additionally, or alternatively, it is also to be noted that, in variousembodiments of the present disclosure, one or more of the NHE-Zinhibiting compounds (monovalent or divalent) detailed herein, whenadministered either alone or in combination with one or more additionalpharmaceutically active compounds or agents (including, for example, afluid-absorbing polymer) to a patient in need thereof, may have aC_(max) of less than about 10 ng/ml, about 7.5 ng/ml, about 5 ng/ml,about 2.5 ng/ml, about 1 ng/ml, or about 0.5 ng/ml, the C_(max) beingfor example within the range of about 1 ng/ml to about 10 ng/ml, orabout 2.5 ng/ml to about 7.5 ng/ml.

Additionally, or alternatively, it is also to be noted that, in variousembodiments of the present disclosure, one or more of the NHE-Zinhibiting compounds (monovalent or divalent) detailed herein, whenadministered either alone or in combination with one or more additionalpharmaceutically active compounds or agents (including, for example, afluid-absorbing polymer) to a patient in need thereof, may have a IC₅₀of less than about 10 μM, about 7.5 μM, about 5 μM, about 2.5 μM, about1 μM, or about 0.5 μM, the IC₅₀ being for example within the range ofabout 1 μM to about 10 μM, or about 2.5 μM to about 7.5 μM.

Additionally, or alternatively, it is also to be noted that, in variousembodiments of the present disclosure, one or more of the NHE-Zinhibiting compounds (monovalent or divalent) detailed herein, whenadministered to a patient in need thereof, may have a ratio ofIC₅₀:C_(max), wherein IC₅₀ and C_(max) are expressed in terms of thesame units, of at least about 10, about 50, about 100, about 250, about500, about 750, or about 1000.

Additionally, or alternatively, it is also to be noted that, in variousembodiments of the present disclosure, wherein one or more of the NHE-Zinhibiting compounds (monovalent or divalent) as detailed herein isorally administered to a patent in need thereof, within the therapeuticrange or concentration, the maximum compound concentration detected inthe serum, defined as C_(max), is lower than the NHE inhibitoryconcentration IC₅₀ of said compound. As previously noted, as usedherein. IC₅₀ is defined as the quantitative measure indicating theconcentration of the compound required to inhibit 50% of theNHE-mediated Na/H antiport activity in a cell based assay.

IV. Pharmaceutical Compositions and Methods of Treatment A. Compositionsand Methods

1. Fluid Retention and/or Salt Overload Disorders

A pharmaceutical composition or preparation that may be used inaccordance with the present disclosure for the treatment of variousdisorders associated with fluid retention and/or salt overload in thegastrointestinal tract (e.g., hypertension, heart failure (inparticular, congestive heart failure), chronic kidney disease, end-stagerenal disease, liver disease and/or peroxisome proliferator-activatedreceptor (PPAR) gamma agonist-induced fluid retention) comprises, ingeneral, the substantially impermeable or substantially systemicallynon-bioavailable NHE-inhibiting compound of the present disclosure, aswell as various other optional components as further detailed hereinbelow (e.g., pharmaceutically acceptable excipients, etc.). Thecompounds utilized in the treatment methods of the present disclosure,as well as the pharmaceutical compositions comprising them, mayaccordingly be administered alone, or as part of a treatment protocol orregiment that includes the administration or use of other beneficialcompounds (as further detailed elsewhere herein). In some particularembodiments, the NHE-inhibiting compound, including any pharmaceuticalcomposition comprising the compound, is administered with afluid-absorbing polymer (as more fully described below).

A “subject” or “mammal” is preferably a human, but can also be an animalin need of treatment with a compound of the disclosure, e.g., companionanimals (e.g., dogs, cats, and the like), farm animals (e.g., cows,pigs, horses and the like) and laboratory animals (e.g., rats, mice,guinea pigs and the like).

Subjects “in need of treatment” with a compound of the presentdisclosure, or subjects “in need of NHE inhibition” include subjectswith diseases and/or conditions that can be treated with substantiallyimpermeable or substantially systemically non-bioavailableNHE-inhibiting compounds, with or without a fluid-absorbing polymer, toachieve a beneficial therapeutic and/or prophylactic result. Abeneficial outcome includes a decrease in the severity of symptoms ordelay in the onset of symptoms, increased longevity and/or more rapid ormore complete resolution of the disease or condition. For example, asubject in need of treatment may be suffering from hypertension; fromsalt-sensitive hypertension which may result from dietary salt intake;from a risk of a cardiovascular disorder (e.g., myocardial infarction,congestive heart failure and the like) resulting from hypertension, fromheart failure (e.g., congestive heart failure) resulting in fluid orsalt overload; from chronic kidney disease resulting in fluid or saltoverload, from end stage renal disease resulting in fluid or saltoverload; from liver disease resulting in fluid or salt overload; fromperoxisome proliferator-activated receptor (PPAR) gamma agonist-inducedfluid retention; or from edema resulting from congestive heart failureor end stage renal disease. In various embodiments, a subject in need oftreatment typically shows signs of hypervolemia resulting from salt andfluid retention that are common features of congestive heart failure,renal failure, liver cirrhosis, gastric ulcers, infectious diarrhea,cancer (colorectal), “leaky gut syndrome”, cystic fibrossigastrointestinal disease, multi-organ failure, microscopic colitis,necrotizing enterocolitis, atopy, food allergy, infections(respiratory), acute inflammation (e.g., sepsis, systemic inflammatoryresponse syndrome), chronic inflammation (e.g., arthritis),obesity-induced metabolic diseases (e.g., nonalcoholic steatohepatitis.Type I diabetes, Type II diabetes, cardiovascular disease), kidneydisease, diabetic kidney disease, cirrhosis, nonalcoholicsteatohepatitis, nonalcoholic fatty acid liver disease, Steatosis,primary sclerosing cholangitis, primary biliary cholangitis, portalhypertension, autoimmune disease (e.g., Type 1 diabetes, ankylosingspondylitis, lupus, alopecia areata, rheumatoid arthritis, polymyalgiarheumatica, fibromyalgia, chronic fatigue syndrome, Sjogren's syndrome,vitiligo, thyroiditis, vasculitis, urticarial (hives), Raynaud'ssyndrome), Schizophrenia, autism spectrum disorders, hepaticencephlopathy, small intestinal bacterial overgrowth, chronicalcoholism, and the like. Fluid retention and salt retention manifestthemselves by the occurrence of shortness of breath, edema, ascites orinterdialytic weight gain. Other examples of subjects that would benefitfrom the treatment are those suffering from congestive heart failure andhypertensive patients and, particularly, those who are resistant totreatment with diuretics, i.e., patients for whom very few therapeuticoptions are available. A subject “in need of treatment” also includes asubject with hypertension, salt-sensitive blood pressure and subjectswith systolic/diastolic blood pressure greater than about 130-139/85-89mm Hg.

In yet other embodiments, the constipation is associated with gastriculcers, infectious diarrhea, cancer (colorectal), “leaky gut syndrome”,cystic fibrossi gastrointestinal disease, multi-organ failure,microscopic colitis, necrotizing enterocolitis, atopy, food allergy,infections (respiratory), acute inflammation (e.g., sepsis, systemicinflammatory response syndrome), chronic inflammation (e.g., arthritis),obesity-induced metabolic diseases (e.g., nonalcoholic steatohepatitis,Type 1 diabetes. Type II diabetes, cardiovascular disease), kidneydisease, diabetic kidney disease, cirrhosis, nonalcoholicsteatohepatitis, nonalcoholic fatty acid liver disease, Steatosis,primary sclerosing cholangitis, primary biliary cholangitis, portalhypertension, autoimmune disease (e.g., Type 1 diabetes, ankylosingspondylitis, lupus, alopecia areata, rheumatoid arthritis, polymyalgiarheumatica, fibromyalgia, chronic fatigue syndrome, Sjogren's syndrome,vitiligo, thyroiditis, vasculitis, urticarial (hives), Raynaud'ssyndrome), Schizophrenia, autism spectrum disorders, hepaticencephlopathy, small intestinal bacterial overgrowth, chronicalcoholism, and the like.

Administration of NHE inhibitors, with or without administration offluid-absorbing polymers, may be beneficial for patients put on“non-added salt” dietary regimen (i.e., 60-100 mmol of Na per day), toliberalize their diet while keeping a neutral or slightly negativesodium balance (i.e., the overall uptake of salt would be equal of lessthan the secreted salt). In that context, “liberalize their diet” meansthat patients treated may add salt to their meals to make the meals morepalatable, or/and diversify their diet with salt-containing foods, thusmaintaining a good nutritional status while improving their quality oflife.

The treatment methods described herein may also help patients with edemaassociated with chemotherapy, pre-menstrual fluid overload andpreeclampsia (pregnancy-induced hypertension).

Accordingly, it is to be noted that the present disclosure is furtherdirected to methods of treatment involving the administration of thecompound of the present disclosure, or a pharmaceutical compositioncomprising such a compound. Such methods may include, for example, amethod for treating hypertension, the method comprising administering tothe patient a substantially impermeable or substantially systemicallynon-bioavailable NHE-inhibiting compound, or a composition comprisingit. The method may be for reducing fluid overload associated with heartfailure (in particular, congestive heart failure), the method comprisingadministering to the patient a substantially impermeable orsubstantially systemically non-bioavailable NHE-inhibiting compound orpharmaceutical composition comprising it. The method may be for reducingfluid overload associated with end stage renal disease, the methodcomprising administering to the patient a substantially impermeable orsubstantially systemically non-bioavailable NHE-inhibiting compound orcomposition comprising it. The method may be for reducing fluid overloadassociated with peroxisome proliferator-activated receptor (PPAR) gammaagonist therapy, the method comprising administering to the patient asubstantially impermeable or substantially systemically non-bioavailableNHE-inhibiting compound or composition comprising it. Additionally, oralternatively, the method may be for decreasing the activity of anintestinal NHE transporter in a patient, the method comprising:administering to the patient a substantially impermeable orsubstantially systemically non-bioavailable NHE-inhibiting compound, ora composition comprising it.

2. Gastrointestinal Tract Disorders

A pharmaceutical composition or preparation that may be used inaccordance with the present disclosure for the treatment of variousgastrointestinal tract disorders, including the treatment or reductionof pain associated with gastrointestinal tract disorders, comprises, ingeneral, any small molecule, which may be monovalent or polyvalent, thatis effective or active as an NHE-inhibitor and that is substantiallyactive in the GI tract, in particular, the substantially impermeable orsubstantially systemically non-bioavailable NHE-inhibiting compound ofthe present disclosure, as well as various other optional components asfurther detailed herein below (e.g., pharmaceutically acceptableexcipients, etc.). The compounds utilized in the treatment methods ofthe present disclosure, as well as the pharmaceutical compositionscomprising them, may accordingly be administered alone, or as part of atreatment protocol or regiment that includes the administration or useof other beneficial compounds (as further detailed elsewhere herein). Insome particular embodiments, the NHE-inhibiting compound, including anypharmaceutical composition comprising the compound, is administered witha fluid-absorbing polymer (as more fully described below).

A “subject” is preferably a human, but can also be an animal in need oftreatment with a compound of the disclosure, e.g., companion animals(e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horsesand the like) and laboratory animals (e.g., rats, mice, guinea pigs andthe like).

Subjects “in need of treatment” with a compound of the presentdisclosure, or subjects “in need of NHE inhibition” include subjectswith diseases and/or conditions that can be treated with substantiallyimpermeable or substantially systemically non-bioavailableNHE-inhibiting compounds, with or without a fluid-absorbing polymer, toachieve a beneficial therapeutic and/or prophylactic result. Abeneficial outcome includes a decrease in the severity of symptoms ordelay in the onset of symptoms, increased longevity and/or more rapid ormore complete resolution of the disease or condition. For example, asubject in need of treatment is suffering from a gastrointestinal tractdisorder; the patient is suffering from a disorder selected from thegroup consisting of: a gastrointestinal motility disorder, irritablebowel syndrome, chronic constipation, chronic idiopathic constipation,chronic constipation occurring in cystic fibrosis patients, chronicconstipation occurring in chronic kidney disease patients,calcium-induced constipation in osteoporotic patients, opioid-inducedconstipation, a functional gastrointestinal tract disorder,gastroesophageal reflux disease, functional heartburn, dyspepsia,functional dyspepsia, non-ulcer dyspepsia, gastroparesis, chronicintestinal pseudo-obstruction, Crohn's disease, ulcerative colitis andrelated diseases referred to as inflammatory bowel syndrome, colonicpseudo-obstruction, and the like.

In various preferred embodiments, the constipation to be treated is:associated with the use of a therapeutic agent; associated with aneuropathic disorder; post-surgical constipation (postoperative ileus);associated with a gastrointestinal tract disorder; idiopathic(functional constipation or slow transit constipation); associated withneuropathic, metabolic or endocrine disorder (e.g., diabetes mellitus,renal failure, hypothyroidism, hyperthyroidism, hypocalcaemia, MultipleSclerosis, Parkinson's disease, spinal cord lesions, neurofibromatosis,autonomic neuropathy, Chagas disease, Hirschsprung's disease or cysticfibrosis, and the like). Constipation may also be the result of surgery(postoperative ileus) or due the use of drugs such as analgesics (e.g.,opioids), antihypertensives, anticonvulsants, antidepressants,antispasmodics and antipsychotics.

Accordingly, it is to be noted that the present disclosure is furtherdirected to methods of treatment involving the administration of thecompound of the present disclosure, or a pharmaceutical compositioncomprising such a compound. Such methods may include, for example, amethod for increasing gastrointestinal motility in a patient, the methodcomprising administering to the patient a substantially non-permeable orsubstantially non-bioavailable NHE-inhibiting compound, or a compositioncomprising it. Additionally, or alternatively, the method may be fordecreasing the activity of an intestinal NHE transporter in a patient,the method comprising: administering to the patient a substantiallynon-permeable or substantially non-bioavailable NHE-inhibiting compound,or a composition comprising it. Additionally, or alternatively, themethod may be for treating a gastrointestinal tract disorder, agastrointestinal motility disorder, irritable bowel syndrome, chroniccalcium-induced constipation in osteoporotic patients, chronicconstipation occurring in cystic fibrosis patients, chronic constipationoccurring in chronic kidney disease patients, a functionalgastrointestinal tract disorder, gastroesophageal reflux disease,functional heartburn, dyspepsia, functional dyspepsia, non-ulcerdyspepsia, gastroparesis, chronic intestinal pseudo-obstruction, colonicpseudo-obstruction, Crohn's disease, ulcerative colitis, inflammatorybowel disease, the method comprising administering an antagonist of theintestinal NHE, and more specifically a substantially non-permeable orsubstantially non-bioavailable NHE-inhibiting compound, or composition,either orally or by rectal suppository. Additionally, or alternatively,the method may be for treating or reducing pain, including visceralpain, pain associated with a gastrointestinal tract disorder or painassociated with some other disorder, the method comprising administeringto a patient a substantially non-permeable or substantiallynon-bioavailable NHE-inhibiting compound, or composition. Additionally,or alternatively, the method may be for treating inflammation, includinginflammation of the gastrointestinal tract, e.g., inflammationassociated with a gastrointestinal tract disorder or infection or someother disorder, the method comprising administering to a patient asubstantially non-permeable or substantially non-bioavailableNHE-inhibiting compound, or composition.

B. Combination Therapies

1. Fluid Retention and/or Salt Overload Disorders

As previously noted, the compounds described herein can be used alone orin combination with other agents. For example, the compounds can beadministered together with a diuretic (i.e., High Ceiling LoopDiuretics, Benzothiadiazide Diuretics, Potassium Sparing Diuretics,Osmotic Diuretics), cardiac glycoside, ACE inhibitor, angiotensin-2receptor antagonist, calcium channel blocker, beta blocker, alphablocker, central alpha agonist, vasodilator, blood thinner,anti-platelet agent, lipid-lowering agent, peroxisomeproliferator-activated receptor (PPAR) gamma agonist agent or compoundor with a fluid-absorbing polymer as more fully described below. Theagent can be covalently attached to a compound described herein or itcan be a separate agent that is administered together with orsequentially with a compound described herein in a combination therapy.

Combination therapy can be achieved by administering two or more agents,e.g., a substantially non-permeable or substantially systemicallynon-bioavailable NHE-inhibiting compound described herein and adiuretic, cardiac glycoside, ACE inhibitor, angiotensin-2 receptorantagonist, calcium channel blocker, beta blocker, alpha blocker,central alpha agonist, vasodilator, blood thinner, anti-platelet agentor compound, each of which is formulated and administered separately, orby administering two or more agents in a single formulation. Othercombinations are also encompassed by combination therapy. For example,two agents can be formulated together and administered in conjunctionwith a separate formulation containing a third agent. While the two ormore agents in the combination therapy can be administeredsimultaneously, they need not be. For example, administration of a firstagent (or combination of agents) can precede administration of a secondagent (or combination of agents) by minutes, hours, days, or weeks.Thus, the two or more agents can be administered within minutes of eachother or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other orwithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other orwithin 2, 3, 4, 5, 6, 7, 8, 9, or weeks of each other. In some caseseven longer intervals are possible. While in many cases it is desirablethat the two or more agents used in a combination therapy be present inwithin the patient's body at the same time, this need not be so.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX—Y—X, X—X—Y, Y—X—Y, Y—Y—X, X—X—Y—Y, etc.

The compounds described herein can be used in combination therapy with adiuretic. Among the useful analgesic agents are, for example: HighCeiling Loop Diuretics [Furosemide (Lasix), Ethacrynic Acid (Edecrin),Bumetanide (Bumex)], Benzothiadiazide Diuretics [Hydrochlorothiazide(Hydrodiuril), Chlorothiazide (Diuril), Clorthalidone (Hygroton),Benzthiazide (Aguapres), Bendroflumethiazide (Naturetin),Methyclothiazide (Aguatensen), Polythiazide (Renese), Indapamide(Lozol), Cyclothiazide (Anhydron), Hydroflumethiazide (Diucardin),Metolazone (Diulo), Quinethazone (Hydromox), Trichlormethiazide(Naqua)], Potassium Sparing Diuretics [Spironolactone (Aldactone),Triamterene (Dyrenium), Amiloride (Midamor)], and Osmotic Diuretics[Mannitol (Osmitrol)]. Diuretic agents in the various classes are knownand described in the literature.

Cardiac glycosides (cardenolides) or other digitalis preparations can beadministered with the compounds of the disclosure in co-therapy. Amongthe useful cardiac glycosides are, for example: Digitoxin (Crystodigin),Digoxin (Lanoxin) or Deslanoside (Cedilanid-D). Cardiac glycosides inthe various classes are described in the literature.

Angiotensin Converting Enzyme Inhibitors (ACE Inhibitors) can beadministered with the compounds of the disclosure in co-therapy. Amongthe useful ACE inhibitors are, for example: Captopril (Capoten),Enalapril (Vasotec), Lisinopril (Prinivil). ACE inhibitors in thevarious classes are described in the literature.

Angiotensin-2 Receptor Antagonists (also referred to as AT₁-antagonistsor angiotensin receptor blockers, or ARB's) can be administered with thecompounds of the disclosure in co-therapy. Among the usefulAngiotensin-2 Receptor Antagonists are, for example: Candesartan(Atacand), Eprosartan (Teveten), Irbesartan (Avapro), Losartan (Cozaar),Telmisartan (Micardis), Valsartan (Diovan). Angiotensin-2 ReceptorAntagonists in the various classes are described in the literature.

Calcium channel blockers such as Amlodipine (Norvasc, Lotrel), Bepridil(Vascor), Diltiazem (Cardizem, Tiazac), Felodipine (Plendil), Nifedipine(Adalat, Procardia), Nimodipine (Nimotop), Nisoldipine (Sular),Verapamil (Calan, Isoptin, Verelan) and related compounds described in,for example, EP 625162B1, U.S. Pat. Nos. 5,364,842, 5,587,454,5,824,645, 5,859,186, 5,994,305, 6,087,091, 6,136,786, WO 93/13128 A1,EP 1336409 A1, EP 835126 A1, EP 835126 B, U.S. Pat. Nos. 5,795,864,5,891,849, 6,054,429, WO 97/01351 A1, the entire contents of which areincorporated herein by reference for all relevant and consistentpurposes, can be used with the compounds of the disclosure.

Beta blockers can be administered with the compounds of the disclosurein co-therapy. Among the useful beta blockers are, for example:Acebutolol (Sectral). Atenolol (Tenormin), Betaxolol (Kerlone),Bisoprolol/hydrochlorothiazide (Ziac), Bisoprolol (Zebeta), Carteolol(Cartrol), Metoprolol (Lopressor, Toprol XL), Nadolol (Corgard),Propranolol (Inderal), Sotalol (Betapace), Timolol (Blocadren). Betablockers in the various classes are described in the literature.

PPAR gamma agonists such as thiazolidinediones (also called glitazones)can be administered with the compounds of the disclosure in co-therapy.Among the useful PPAR agonists are, for example: rosiglitazone(Avandia), pioglitazone (Actos) and rivoglitazone.

Aldosterone antagonists can be administered with the compounds of thedisclosure in co-therapy. Among the useful Aldosterone antagonists are,for example: eplerenone, spironolactone, and canrenone.

Alpha blockers can be administered with the compounds of the disclosurein co-therapy. Among the useful Alpha blockers are, for example:Doxazosin mesylate (Cardura), Prazosin hydrochloride (Minipress).Prazosin and polythiazide (Minizide), Terazosin hydrochloride (Hytrin).Alpha blockers in the various classes are described in the literature.

Central alpha agonists can be administered with the compounds of thedisclosure in co-therapy. Among the useful Central alpha agonists are,for example: Clonidine hydrochloride (Catapres), Clonidine hydrochlorideand chlorthalidone (Clorpres. Combipres), Guanabenz Acetate (Wytensin),Guanfacine hydrochloride (Tenex), Methyldopa (Aldomet), Methyldopa andchlorothiazide (Aldochlor), Methyldopa and hydrochlorothiazide(Aldoril). Central alpha agonists in the various classes are describedin the literature.

Vasodilators can be administered with the compounds of the disclosure inco-therapy. Among the useful vasodilators are, for example: Isosorbidedinitrate (Isordil), Nesiritide (Natrecor), Hydralazine (Apresoline),Nitrates/nitroglycerin, Minoxidil (Loniten). Vasodilators in the variousclasses are described in the literature.

Blood thinners can be administered with the compounds of the disclosurein co-therapy. Among the useful blood thinners are, for example:Warfarin (Coumadin) and Heparin. Blood thinners in the various classesare described in the literature.

Anti-platelet agents can be administered with the compounds of thedisclosure in co-therapy. Among the useful anti-platelet agents are, forexample: Cyclooxygenase inhibitors (Aspirin), Adenosine diphosphate(ADP) receptor inhibitors [Clopidogrel (Plavix), Ticlopidine (Ticlid)],Phosphodiesterase inhibitors [Cilostazol (Pletal)], GlycoproteinIIB/IIIA inhibitors [Abciximab (ReoPro), Eptifibatide (Integrilin),Tirofiban (Aggrastat), Defibrotide], Adenosine reuptake inhibitors[Dipyridamole (Persantine)]. Anti-platelet agents in the various classesare described in the literature.

Lipid-lowering agents can be administered with the compounds of thedisclosure in co-therapy. Among the useful lipid-lowering agents are,for example: Statins (HMG CoA reductase inhibitors), [Atorvastatin(Lipitor), Fluvastatin (Lescol), Lovastatin (Mevacor, Altoprev),Pravastatin (Pravachol), Rosuvastatin Calcium (Crestor), Simvastatin(Zocor)], Selective cholesterol absorption inhibitors [ezetimibe(Zetia)], Resins (bile acid sequestrant or bile acid-binding drugs)[Cholestyramine (Questran, Questran Light, Prevalite, Locholest,Locholest Light), Colestipol (Colestid). Colesevelam Hcl (WelChol)],Fibrates (Fibric acid derivatives) [Gemfibrozil (Lopid), Fenofibrate(Antara, Lofibra, Tricor, and Triglide), Clofibrate (Atromid-S)], Niacin(Nicotinic acid). Lipid-lowering agents in the various classes aredescribed in the literature.

The compounds of the disclosure can be used in combination with peptidesor peptide analogs that activate the Guanylate Cyclase-receptor in theintestine and results in elevation of the intracellular secondmessenger, or cyclic guanosine monophosphate (cGMP), with increasedchloride and bicarbonate secretion into the intestinal lumen andconcomitant fluid secretion. Example of such peptides are Linaclotide(MD-1100 Acetate), endogenous hormones guanylin and uroguanylin andenteric bacterial peptides of the heat stable enterotoxin family (STpeptides) and those described in U.S. Pat. Nos. 5,140,102, 5,489,670,5,969,097, WO 2006/001931A2, WO 2008/002971A2, WO 2008/106429A2, US2008/0227685A1 and U.S. Pat. No. 7,041,786, the entire contents of whichare incorporated herein by reference for all relevant and consistentpurposes.

The compounds of the disclosure can be used in combination with type-2chloride channel agonists, such as Amitiza (Lubiprostone) and otherrelated compounds described in U.S. Pat. No. 6,414,016, the entirecontents of which are incorporated herein by reference for all relevantand consistent purposes.

The compounds of the disclosure can be used in combination with P2Y2receptor agonists, such as those described in EP 1196396B1 and U.S. Pat.No. 6,624,150, the entire contents of which are incorporated herein byreference for all relevant and consistent purposes.

Other agents include natriuretic peptides such as nesiritide, arecombinant form of brain-natriuretic peptide (BNP) and anatrial-natriuretic peptide (ANP). Vasopressin receptor antagonists suchas tolvaptan and conivaptan may be co-administered as well as phosphatebinders such as renagel, renleva, phoslo and fosrenol. Other agentsinclude phosphate transport inhibitors (as described in U.S. Pat. Nos.4,806,532; 6,355,823; 6,787,528; 7,119,120; 7,109,184; U.S. Pat. Pub.No. 2007/021509; 2006/0280719; 2006/0217426: International Pat. Pubs. WO2001/005398, WO 2001/087294, WO 2001/082924, WO 2002/028353, WO2003/048134, WO 2003/057225, WO2003/080630, WO 2004/085448, WO2004/085382; European Pat. Nos. 1465638 and 1485391; and JP Patent No.2007131532, or phosphate transport antagonists such as Nicotinamide.

2. Gastrointestinal Tract Disorders

As previously noted, the compounds described herein can be used alone orin combination with other agents. For example, the compounds can beadministered together with an analgesic peptide or compound. Theanalgesic peptide or compound can be covalently attached to a compounddescribed herein or it can be a separate agent that is administeredtogether with or sequentially with a compound described herein in acombination therapy.

Combination therapy can be achieved by administering two or more agents,e.g., a substantially non-permeable or substantially non-bioavailableNHE-inhibiting compound described herein and an analgesic peptide orcompound, each of which is formulated and administered separately, or byadministering two or more agents in a single formulation. Othercombinations are also encompassed by combination therapy. For example,two agents can be formulated together and administered in conjunctionwith a separate formulation containing a third agent. While the two ormore agents in the combination therapy can be administeredsimultaneously, they need not be. For example, administration of a firstagent (or combination of agents) can precede administration of a secondagent (or combination of agents) by minutes, hours, days, or weeks.Thus, the two or more agents can be administered within minutes of eachother or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other orwithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other orwithin 2, 3, 4, 5, 6, 7, 8, 9, or weeks of each other. In some caseseven longer intervals are possible. While in many cases it is desirablethat the two or more agents used in a combination therapy be present inwithin the patient's body at the same time, this need not be so.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX—Y—X, X—X—Y, Y—X—Y, Y—Y—X, X—X—Y—Y, etc.

The compounds described herein can be used in combination therapy withan analgesic agent, e.g., an analgesic compound or an analgesic peptide.The analgesic agent can optionally be covalently attached to a compounddescribed herein. Among the useful analgesic agents are, for example: Cachannel blockers, 5HT3 agonists (e.g., MCK-733), 5HT4 agonists (e.g.,tegaserod, prucalopride), and 5HT1 receptor antagonists, opioid receptoragonists (loperamide, fedotozine, and fentanyl), NK1 receptorantagonists, CCK receptor agonists (e.g., loxiglumide), NK1 receptorantagonists, NK3 receptor antagonists, norepinephrine-serotonin reuptakeinhibitors (NSR1), vanilloid and cannabanoid receptor agonists, andsialorphin. Analgesics agents in the various classes are described inthe literature.

Opioid receptor antagonists and agonists can be administered with thecompounds of the disclosure in co-therapy or linked to the compound ofthe disclosure, e.g., by a covalent bond. For example, opioid receptorantagonists such as naloxone, naltrexone, methyl nalozone, nalmefene,cypridime, beta funaltrexamine, naloxonazine, naltrindole, andnor-binaltorphimine are thought to be useful in the treatment ofopioid-induced constipaption (OIC). It can be useful to formulate opioidantagonists of this type in a delayed or sustained release formulation,such that initial release of the antagonist is in the mid to distalsmall intestine and/or ascending colon. Such antagonists are describedin U.S. Pat. No. 6,734,188 (WO 01/32180 A2), the entire contents ofwhich are incorporated herein by reference for all relevant andconsistent purposes. Enkephalin pentapeptide (HOE825:Tyr-D-Lys-Gly-Phe-L-homoserine) is an agonist of the μ- and γ-opioidreceptors and is thought to be useful for increasing intestinal motility(Eur. J. Pharm., 219:445, 1992), and this peptide can be used inconjunction with the compounds of the disclosure. Also useful istrimebutine which is thought to bind to mu/delta/kappa opioid receptorsand activate release of motilin and modulate the release of gastrin,vasoactive intestinal peptide, gastrin and glucagons. K-opioid receptoragonists such as fedotozine, ketocvclazocine, and compounds described inUS 2005/0176746 (WO 03/097051 A2), the entire contents of which areincorporated herein by reference for all relevant and consistentpurposes, can be used with or linked to the compounds of the disclosure.In addition, μ-opioid receptor agonists, such as morphine,diphenyloxylate, frakefamide (H-Tyr-D-Ala-Phe(F)-Phe-NH₂; disclosed inWO 01/019849 A1, the entire contents of which are incorporated herein byreference for all relevant and consistent purposes) and loperamide canbe used.

Tyr-Arg (kyotorphin) is a dipeptide that acts by stimulating the releaseof met-enkephalins to elicit an analgesic effect (J. Biol. Chem.262:8165, 1987). Kyotorphin can be used with or linked to the compoundsof the disclosure. CCK receptor agonists such as caerulein fromamphibians and other species are useful analgesic agents that can beused with or linked to the compounds of the disclosure.

Conotoxin peptides represent a large class of analgesic peptides thatact at voltage gated Ca channels, NMDA receptors or nicotinic receptors.These peptides can be used with or linked to the compounds of thedisclosure.

Peptide analogs of thymulin (U.S. Pat. No. 7,309,690 or FR 2830451, theentire contents of which are incorporated herein by reference for allrelevant and consistent purposes) can have analgesic activity and can beused with or linked to the compounds of the disclosure.

CCK (CCKa or CCKb) receptor antagonists, including loxiglumide anddexloxiglumide (the R-isomer of loxiglumide) (U.S. Pat. No. 5,130,474 orWO 88/05774, the entire contents of which are incorporated herein byreference for all relevant and consistent purposes) can have analgesicactivity and can be used with or linked to the compounds of thedisclosure.

Other useful analgesic agents include 5-HT4 agonists such astegaserod/zelnorm and lirexapride. Such agonists are described in:EP1321142 A1, WO 03/053432A1, EP 505322 A1, EP 505322 B1, EP 507672 A1.EP 507672 B1, U.S. Pat. Nos. 5,510,353 and 5,273,983, the entirecontents of which are incorporated herein by reference for all relevantand consistent purposes.

Calcium channel blockers such as ziconotide and related compoundsdescribed in, for example, EP 625162B1, U.S. Pat. Nos. 5,364,842,5,587,454, 5,824,645, 5,859,186, 5,994,305, 6,087,091, 6,136,786, WO93/13128 A1, EP 1336409 A1, EP 835126 A1, EP 835126 B1, U.S. Pat. Nos.5,795,864, 5,891,849, 6,054,429, WO 97/01351 A1, the entire contents ofwhich are incorporated herein by reference for all relevant andconsistent purposes, can be used with or linked to the compounds of thedisclosure.

Various antagonists of the NK-1, NK-2, and NK-3 receptors (for a reviewsee Giardina et al. 2003 Drugs 6:758) can be can be used with or linkedto the compounds of the disclosure.

NK1 receptor antagonists such as: aprepitant (Merck & Co Inc),vofopitant, ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La Roche Ltd),SR-14033 and related compounds described in, for example, EP 873753 A1,U.S. 20010006972 A1, U.S. 20030109417 A1, WO 01/52844 A1, the entirecontents of which are incorporated herein by reference for all relevantand consistent purposes, can be used with or linked to the compounds ofthe disclosure.

NK-2 receptor antagonists such as nepadutant (Menarini Ricerche SpA),saredutant (Sanofi-Synthelabo), SR-144190 (Sanofi-Synthelabo) andUK-290795 (Pfizer Inc) can be used with or linked to the compounds ofthe disclosure.

NK3 receptor antagonists such as osanetant (Sanofi-Synthelabo),talnetant and related compounds described in, for example, WO 02/094187A2, EP 876347 A1. WO 97/21680 A1, U.S. Pat. No. 6,277,862, WO 98/11090,WO 95/28418, WO 97/19927, and Boden et al. (J Med. Chem. 39:1664-75,1996), the entire contents of which are incorporated herein by referencefor all relevant and consistent purposes, can be used with or linked tothe compounds of the disclosure.

Norepinephrine-serotonin reuptake inhibitors such as milnacipran andrelated compounds described in WO 03/077897 A1, the entire contents ofwhich are incorporated herein by reference for all relevant andconsistent purposes, can be used with or linked to the compounds of thedisclosure.

Vanilloid receptor antagonists such as arvanil and related compoundsdescribed in WO 01/64212 A1, the entire contents of which areincorporated herein by reference for all relevant and consistentpurposes, can be used with or linked to the compounds of the disclosure.

The compounds can be used in combination therapy with aphosphodiesterase inhibitor (examples of such inhibitors can be found inU.S. Pat. No. 6,333,354, the entire contents of which are incorporatedherein by reference for all relevant and consistent purposes).

The compounds can be used alone or in combination therapy to treatdisorders associated with chloride or bicarbonate secretion that maylead to constipation, e.g., Cystic Fibrosis.

The compounds can also or alternatively be used alone or in combinationtherapy to treat calcium-induced constipation effects. Constipation iscommonly found in the geriatric population, particularly patients withosteoporosis who have to take calcium supplements. Calcium supplementshave shown to be beneficial in ostoporotic patients to restore bonedensity but compliance is poor because of constipation effectsassociated therewith.

The compounds of the current disclosure have can be used in combinationwith an opioid. Opioid use is mainly directed to pain relief, with anotable side-effect being GI disorder, e.g. constipation. These agentswork by binding to opioid receptors, which are found principally in thecentral nervous system and the gastrointestinal tract. The receptors inthese two organ systems mediate both the beneficial effects, and theundesirable side effects (e.g. decrease of gut motility and ensuingconstipation). Opioids suitable for use typically belong to one of thefollowing exemplary classes: natural opiates, alkaloids contained in theresin of the opium poppy including morphine, codeine and thebaine;semi-synthetic opiates, created from the natural opioids, such ashydromorphone, hydrocodone, oxycodone, oxymorphone, desomorphine,diacetylmorphine (Heroin), nicomorphine, dipropanoylmorphine,benzylmorphine and ethylmorphine; fully synthetic opioids, such asfentanyl, pethidine, methadone, tramadol and propoxyphene; endogenousopioid peptides, produced naturally in the body, such as endorphins,enkephalins, dynorphins, and endomorphins.

The compound of the disclosure can be used alone or in combinationtherapy to alleviate GI disorders encountered with patients with renalfailure (stage 3-5). Constipation is the second most reported symptom inthat category of patients (Murtagh et al., 2006: Murtagh et al., 2007a;Murtagh et al., 2007b). Without being held by theory, it is believedthat kidney failure is accompanied by a stimulation of intestinal Nare-absorption (Hatch and Freel, 2008). A total or partial inhibition ofsuch transport by administration of the compounds of the disclosure canhave a therapeutic benefit to improve GI transit and relieve abdominalpain. In that context, the compounds of the disclosure can be used incombination with Angiotensin-modulating agents: Angiotensin ConvertingEnzyme (ACE) inhibitors (e.g. captopril, enalopril, lisinopril,ramipril) and Angiotensin II receptor antagonist therapy (also referredto as ATI-antagonists or angiotensin receptor blockers, or ARB's);diuretics such as loop diuretics (e.g. furosemide, bumetanide), Thiazidediuretics (e.g. hydrochlorothiazide, chlorthalidone, chlorthiazide) andpotassium-sparing diuretics: amiloride; beta blockers: bisoprolol,carvedilol, nebivolol and extended-release metoprolol; positiveinotropes: Digoxin, dobutamine; phosphodiesterase inhibitors such asmilrinone; alternative vasodilators: combination of isosorbidedinitrate/hydralazine; aldosterone receptor antagonists; spironolactone,eplerenone; natriuretic peptides: Nesiritide, a recombinant form ofbrain-natriuretic peptide (BNP), atrial-natriuretic peptide (ANP);vasopressin receptor antagonists: Tolvaptan and conivaptan; phosphatebinder (Renagel, Renleva, Phoslo, Fosrenol); phosphate transportinhibitor such as those described in U.S. Pat. Nos. 4,806,532,6,355,823, 6,787,528, WO 2001/005398, WO 2001/087294, WO 2001/082924, WO2002/028353, WO 2003/048134, WO 2003/057225, U.S. Pat. No. 7,119,120, EP1465638, US Appl. 2007/021509, WO 2003/080630, U.S. Pat. No. 7,109,184,US Appl. 2006/0280719, EP 1485391, WO 2004/085448, WO 2004/085382, USAppl. 2006/0217426, JP 2007/131532, the entire contents of which areincorporated herein by reference for all relevant and consistentpurposes, or phosphate transport antagonist (Nicotinamide).

The compounds of the disclosure can be used in combination with peptidesor peptide analogs that activate the Guanylate Cyclase-receptor in theintestine and results in elevation of the intracellular secondmessenger, or cyclic guanosine monophosphate (cGMP), with increasedchloride and bicarbonate secretion into the intestinal lumen andconcomitant fluid secretion. Example of such peptides are Linaclotide(MD-1100 Acetate), endogenous hormones guanylin and uroguanylin andenteric bacterial peptides of the heat stable enterotoxin family (STpeptides) and those described in U.S. Pat. Nos. 5,140,102, 5,489,670,5,969,097, WO 2006/001931A2, WO 2008/002971A2, WO 2008/106429A2, US2008/0227685A1 and U.S. Pat. No. 7,041,786, the entire contents of whichare incorporated herein by reference for all relevant and consistentpurposes.

The compounds of the disclosure can be used in combination with type-2chloride channel agonists, such as Amitiza (Lubiprostone) and otherrelated compounds described in U.S. Pat. No. 6,414,016, the entirecontents of which are incorporated herein by reference for all relevantand consistent purposes.

The compounds of the disclosure can be used in combination with P2Y2receptor agonists, such as those described in EP 1196396B1 and U.S. Pat.No. 6,624,150, the entire contents of which are incorporated herein byreference for all relevant and consistent purposes.

The compounds of the disclosure can be used in combination with laxativeagents such as bulk-producing agents, e.g. psyllium husk (Metamucil),methylcellulose (Citrucel), polycarbophil, dietary fiber, apples, stoolsofteners/surfactant such as docusate (Colace, Diocto); hydrating agents(osmotics), such as dibasic sodium phosphate, magnesium citrate,magnesium hydroxide (Milk of magnesia), magnesium sulfate (which isEpsom salt), monobasic sodium phosphate, sodium biphosphate;hyperosmotic agents: glycerin suppositories, sorbitol, lactulose, andpolyethylene glycol (PEG). The compounds of the disclosure can be alsobe used in combination with agents that stimulate gut peristalsis, suchas Bisacodyl tablets (Dulcolax), Casanthranol, Senna and Aloin, fromAloe Vera.

In one embodiment, the compounds of the disclosure accelerategastrointestinal transit, and more specifically in the colon, withoutsubstantially affecting the residence time in the stomach, i.e. with nosignificant effect on the gastric emptying time. Even more specificallythe compounds of the invention restore colonic transit without theside-effects associated with delayed gastric emptying time, such asnausea. The GI and colonic transit are measured in patients usingmethods reported in, for example: Burton D D, Camilleri M. Mullan B P,et al., J. Nucl. Med., 1997; 38:1807-1810; Cremonini F. Mullan B P,Camilleri M, et al., Aliment. Pharmacol. Ther., 2002; 16:1781-1790;Camilleri M. Zinsmeister A R, Gastroenterology, 1992; 103:36-42: BourasE P, Camilleri M, Burton D D, et al., Gastroenterology, 2001;120:354-360: Coulie B, Szarka L A, Camilleri M, et al.,Gastroenterology, 2000; 119:41-50; Prather C M, Camilleri M, ZinsmeisterA R, et al., Gastroenterology, 2000; 118:463-468: and, Camilleri M,McKinzie S, Fox J, et al., Clin. Gastroenterol. Hepatol., 2004;2:895-904.

C. Polymer Combination Therapy

The NHE-inhibiting compounds described therein may be administered topatients in need thereof in combination with a fluid-absorbing polymer(“FAP”). The intestinal fluid-absorbing polymers useful foradministration in accordance with embodiments of the present disclosuremay be administered orally in combination with non-absorbableNHE-inhibitors (e.g., a NHE-3 inhibitor) to absorb the intestinal fluidresulting from the action of the sodium transport inhibitors. Suchpolymers swell in the colon and bind fluid to impart a consistency tostools that is acceptable for patients. The fluid-absorbing polymersdescribed herein may be selected from polymers with laxative properties,also referred to as bulking agents (i.e., polymers that retain some ofthe intestinal fluid in the stools and impart a higher degree ofhydration in the stools and facilitate transit). The fluid-absorbingpolymers may also be optionally selected from pharmaceutical polymerswith anti-diarrhea function, i.e., agents that maintain some consistencyto the stools to avoid watery stools and potential incontinence.

The ability of the polymer to maintain a certain consistency in stoolswith a high content of fluid can be characterized by its “water holdingpower.” Wenzl et al. (in Determinants of decreased fecal consistency inpatients with diarrhea; Gastroenterology, v. 108, no. 6, p. 1729-1738(1995)) studied the determinants that control the consistency of stoolsof patients with diarrhea and found that they were narrowly correlatedwith the water holding power of the feces. The water holding power isdetermined as the water content of given stools to achieve a certainlevel of consistency (corresponding to “formed stool” consistency) afterthe reconstituted fecal matter has been centrifuged at a certain gnumber. Without being held to any particular theory, has been found thatthe water holding power of the feces is increased by ingestion ofcertain polymers with a given fluid absorbing profile. Morespecifically, it has been found that the water-holding power of saidpolymers is correlated with their fluid absorbancy under load (AUL);even more specifically the AUL of said polymers is greater than 15 g ofisotonic fluid/g of polymer under a static pressure of 5 kPa, even morepreferably under a static pressure of 10 kPa.

The FAP utilized in the treatment method of the present disclosurepreferably has a AUL of at least about 10 g, about 15 g, about 20 g,about 25 g or more of isotonic fluid/g of polymer under a staticpressure of about 5 kPa and preferably about 10 kPA, and may have afluid absorbency of about 20 g, about 25 g or more, as determined usingmeans generally known in the art. Additionally or alternatively, the FAPmay impart a minimum consistency to fecal matter and, in someembodiments, a consistency graded as “soft” in the scale described inthe test method below, when fecal non water-soluble solid fraction isfrom 10% to 20%, and the polymer concentration is from 1% to 5% of theweight of stool. The determination of the fecal non water-soluble solidfraction of stools is described in Wenz et al. The polymer may beuncharged or may have a low charge density (e.g., 1-2 meq/gr).Alternatively or in addition, the polymer may be delivered directly tothe colon using known delivery methods to avoid premature swelling inthe esophagus.

In one embodiment of the present disclosure, the FAP is a“superabsorbent” polymer (i.e., a lightly crosslinked, partiallyneutralized polyelectrolyte hydrogel similar to those used in babydiapers, feminine hygiene products, agriculture additives, etc.).Superabsorbent polymers may be made of a lightly crosslinkedpolyacrylate hydrogel. The swelling of the polymer is driven essentiallyby two effects: (i) the hydration of the polymer backbone and entropy ofmixing and (ii) the osmotic pressure arising from the counter-ions(e.g., Na ions) within the gel. The gel swelling ratio at equilibrium iscontrolled by the elastic resistance inherent to the polymer network andby the chemical potential of the bathing fluid, i.e., the gel willde-swell at higher salt concentration because the background electrolytewill reduce the apparent charge density on the polymer and will reducethe difference of free ion concentrations inside and outside the gelthat drives osmotic pressure. The swelling ratio SR (g of fluid per g ofdry polymer and synonymously “fluid absorbency”) may vary from 1000 inpure water down to 30 in 0.9% NaCl solution representative ofphysiological saline (i.e., isotonic). SR may increase with the degreeof neutralization and may decrease with the crosslinking density. SRgenerally decreases with an applied load with the extent of reductiondependent on the strength of the gel, i.e., the crosslinking density.The salt concentration within the gel, as compared with the externalsolution, may be lower as a result of the Donnan effect due to theinternal electrical potential.

The fluid-absorbing polymer may include crosslinked polyacrylates whichare fluid absorbent such as those prepared from α,β-ethylenicallyunsaturated monomers, such as monocarboxylic acids, polycarboxylicacids, acrylamide and their derivatives. These polymers may haverepeating units of acrylic acid, methacrylic acid, metal salts ofacrylic acid, acrylamide, and acrylamide derivatives (such as2-acrylamido-2-methylpropanesulfonic acid) along with variouscombinations of such repeating units as copolymers. Such derivativesinclude acrylic polymers which include hydrophilic grafts of polymerssuch as polyvinyl alcohol. Examples of suitable polymers and processes,including gel polymerization processes, for preparing such polymers aredisclosed in U.S. Pat. Nos. 3,997,484, 3,926,891; 3,935,099; 4,090,013;4,093,776; 4,340,706; 4,446,261; 4,683,274; 4,459,396; 4,708,997;4,076,663; 4,190,562; 4,286,082; 4,857,610; 4,985,518; 5,145,906,5,629,377 and 6,908,609 which are incorporated herein by reference forall relevant and consistent purposes (in addition, see Buchholz, F. L.and Graham, A. T., “Modem Superabsorbent Polymer Technology,” John Wiley& Sons (1998), which is also incorporated herein by reference for allrelevant and consistent purposes). A class of preferred polymers fortreatment in combination with NHE-inhibitors is polyelectrolytes.

The degree of crosslinking can vary greatly depending upon the specificpolymer material; however, in most applications the subjectsuperabsorbent polymers are only lightly crosslinked, that is, thedegree of crosslinking is such that the polymer can still absorb over 10times its weight in physiological saline (i.e., 0.9% saline). Forexample, such polymers typically include less than about 0.2 mole %crosslinking agent.

In some embodiments, the FAP's utilized for treatment are CalciumCarbophil (Registry Number: 9003-97-8, also referred as Carbopol EX-83),and Carpopol 934P.

In some embodiments, the fluid-absorbing polymer is prepared by highinternal phase emulsion (“HIPE”) processes. The HIPE process leads topolymeric foam slabs with a very large porous fraction of interconnectedlarge voids (about 100 microns) (i.e., open-cell structures). Thistechnique produces flexible and collapsible foam materials withexceptional suction pressure and fluid absorbency (see U.S. Pat. Nos.5,650,222; 5,763,499 and 6,107,356, which are incorporated herein forall relevant and consistent purposes). The polymer is hydrophobic and,therefore, the surface should be modified so as to be wetted by theaqueous fluid. This is accomplished by post-treating the foam materialby a surfactant in order to reduce the interfacial tension. Thesematerials are claimed to be less compliant to loads, i.e., less prone tode-swelling under static pressure.

In some embodiments, fluid-absorbing gels are prepared by aqueous freeradical polymerization of acrylamide or a derivative thereof, acrosslinker (e.g., methylene-bis-acrylamide) and a free radicalinitiator redox system in water. The material is obtained as a slab.Typically the swelling ratio of crosslinked polyacrylamide at lowcrosslinking density (e.g., 2%-4% expressed as weight % ofmethylene-bis-acrylamide) is between 25 and 40 (F. Horkay,Macromolecules, 22. pp. 2007-09 (1989)). The swelling properties ofthese polymers have been extensively studied and are essentially thesame of those of crosslinked polyacrylic acids at high saltconcentration. Under those conditions, the osmotic pressure is null dueto the presence of counter-ions and the swelling is controlled by thefree energy of mixing and the network elastic energy. Stateddifferently, a crosslinked polyacrylamide gel of same crosslink densityas a neutralized polyacrylic acid will exhibit the same swelling ratio(i.e., fluid absorbing properties) and it is believed the same degree ofdeswelling under pressure, as the crosslinked polyelectrolyte at highsalt content (e.g., 1 M). The properties (e.g., swelling) of neutralhydrogels will not be sensitive to the salt environment as long as thepolymer remains in good solvent conditions. Without being held to anyparticular theory, it is believed that the fluid contained within thegel has the same salt composition than the surrounding fluid (i.e.,there is no salt partitioning due to Donnan effect).

Another subclass of fluid-absorbing polymers that may be utilized ishydrogel materials that include N-alkyl acrylamide polymers (e.g.,N-isopropylacrylamide (NIPAM)). The corresponding aqueous polyNIPAMhydrogel shows a temperature transition at about 35° C. Above thistemperature the hydrogel may collapse. The mechanism is generallyreversible and the gel re-swells to its original swelling ratio when thetemperature reverts to room temperature. This allows production ofnanoparticles by emulsion polymerization (R. Pelton, Advances in Colloidand Interface Science, 85, pp. 1-33, (2000)). The swellingcharacteristics of poly-NIPAM nanoparticles below the transitiontemperature have been reported and are similar to those reported forbulk gel of polyNIPAM and equivalent to those found for polyacrylamide(i.e. 30-50 g/g) (W. McPhee, Journal of Colloid and Interface Science,156. pp. 24-30 (1993); and, K. Oh, Journal of Applied Polymer Science,69, pp. 109-114 (1997)).

In some embodiments, the FAP utilized for treatment in combination witha NHE-inhibitor is a superporous gel that may delay the emptying of thestomach for the treatment of obesity (J. Chen, Journal of ControlledRelease, 65, pp. 73-82 (2000), or to deliver proteins.Polyacrylate-based SAP's with a macroporous structure may also be used.Macroporous SAP and superporous gels differ in that the porous structureremains almost intact in the dry state for superporous gels, butdisappears upon drying for macroporous SAP's. The method of preparationis different although both methods use a foaming agent (e.g., carbonatesalt that generates CO₂ bubbles during polymerization). Typical swellingratios, SR, of superporous materials are around 10. Superporous gelskeep a large internal pore volume in the dry state.

Macroporous hydrogels may also be formed using a method whereby polymerphase separation in induced by a non-solvent. The polymer may bepoly-NIPAM and the non-solvent utilized may be glucose (see, e.g., Z.Zhang, J. Org. Chem., 69, 23 (2004)) or NaCl (see, e.g., Cheng et al.,Journal of Biomedical Materials Research—Part A, Vol. 67, Issue 1, 1Oct. 2003, Pages 96-103). The phase separation induced by the presenceof NaCl leads to an increase in swelling ratio. These materials arepreferred if the swelling ratio of the material, SR, is maintained insalt isotonic solution and if the gels do not collapse under load. Thetemperature of “service” should be shifted beyond body temperature, e.g.by diluting NIPAM in the polymer with monomer devoid of transitiontemperature phenomenon.

In some embodiments, the fluid-absorbing polymer may be selected fromcertain naturally-occurring polymers such as those containingcarbohydrate moieties. In a preferred embodiment, suchcarbohydrate-containing hydrogels are non-digestible, have a lowfraction of soluble material and a high fraction of gel-formingmaterials. In some embodiments, the fluid-absorbing polymer is selectedfrom xanthan, guar, wellan, hemicelluloses, alkyl-cellulose,hydro-alkyl-cellulose, carboxy-alkyl-cellulose, carrageenan, dextran,hyaluronic acid and agarose. In a preferred embodiment, the gel formingpolymer is psyllium. Psyllium (or “ispaghula”) is the common name usedfor several members of the plant genus Plantago whose seeds are usedcommercially for the production of mucilage. Most preferably, thefluid-absorbing polymer is in the gel-forming fraction of psyllium,i.e., a neutral saccharide copolymer of arabinose (25%) and xylose (75%)as characterized in (J. Marlett, Proceedings of the Nutrition Society,62, pp. 2-7-209 (2003); and, M. Fischer, Carbohydrate Research, 339,2009-2012 (2004)), and further described in U.S. Pat. Nos. 6,287,609;7,026,303; 5,126,150; 5,445,831; 7,014,862; 4,766,004; 4,999,200, eachof which is incorporated herein for all relevant and consistentpurposes, and over-the-counter psyllium-containing agents such as thosemarketed under the name Metamucil (The Procter and Gamble company).Preferably the a psyllium-containing dosage form is suitable forchewing, where the chewing action disintegrates the tablet into smaller,discrete particles prior to swallowing but which undergoes minimalgelling in the mouth, and has acceptable mouthfeel and good aestheticsas perceived by the patient.

The psyllium-containing dosage form includes physically discrete unitsuitable as a unitary dosage for human subjects and other mammals, eachcontaining a predetermined quantity of active material (e.g. thegel-forming polysaccharide) calculated to produce the desiredtherapeutic effect. Solid oral dosage forms that are suitable for thepresent compositions include tablets, pills, capsules, lozenges,chewable tablets, troches, cachets, pellets, wafer and the like.

In some embodiments, the FAP is a polysaccharide particle wherein thepolysaccharide component includes xylose and arabinose. The ratio of thexylose to the arabinose may be at least about 3:1 by weight, asdescribed in U.S. Pat. Nos. 6,287,609; 7,026,303 and 7,014,862, each ofwhich is incorporated herein for all relevant and consistent purposes.

The fluid-absorbing polymers described herein may be used in combinationwith the NHE-inhibiting compounds or a pharmaceutical compositioncontaining the compound. The NHE inhibitor and the FAP may also beadministered with other agents including those described under theheading “Combination Therapies” without departing from the scope of thepresent disclosure. As described above, the NHE inhibitor may beadministered alone without use of a fluid-absorbing polymer to resolvesymptoms without eliciting significant diarrhea or fecal fluid secretionthat would require the co-administration of a fluid-absorbing polymer.

The fluid-absorbing polymers described herein may be selected so as tonot induce any substantial interaction with the NHE-inhibiting compoundsor a pharmaceutical composition containing the compound. As used herein,“no substantial interaction” generally means that the co-administrationof the FAP polymer would not substantially alter (i.e., neithersubstantially decrease nor substantially increase) the pharmacologicalproperty of the NHE-inhibiting compounds administered alone. Forexample, FAPs containing negatively charged functionality, such ascarboxylates, sulfonates, and the like, may potentially interactionically with positively charged NHE inhibitors, preventing theinhibitor from reaching its pharmacological target. In addition, it maybe possible that the shape and arrangement of functionality in a FAPcould act as a molecular recognition element, and sequestor NHEinhibitors via “host-guest” interactions via the recognition of specifichydrogen bonds and/or hydrophobic regions of a given inhibitor.Accordingly, in various embodiments of the present disclosure, the FAPpolymer may be selected, for co-administration or use with a compound ofthe present disclosure, to ensure that (i) it does not ionicallyinteract with or bind with the compound of the present disclosure (bymeans of, for example, a moiety present therein possessing a chargeopposite that of a moiety in the compound itself), and/or (ii) it doesnot possess a charge and/or structural conformation (or shape orarrangement) that enables it to establish a “host-guest” interactionwith the compound of the present disclosure (by means of, for example, amoiety present therein that may act as a molecular recognition elementand sequester the NHE inhibitor or inhibiting moiety of the compound).

D. Dosage

It is to be noted that, as used herein, an “effective amount” (or“pharmaceutically effective amount”) of a compound disclosed herein, isa quantity that results in a beneficial clinical outcome of thecondition being treated with the compound compared with the absence oftreatment. The amount of the compound or compounds administered willdepend on the degree, severity, and type of the disease or condition,the amount of therapy desired, and the release characteristics of thepharmaceutical formulation. It will also depend on the subject's health,size, weight, age, sex and tolerance to drugs. Typically, the compoundis administered for a sufficient period of time to achieve the desiredtherapeutic effect.

In embodiments wherein both an NHE-inhibitor compound and afluid-absorbing polymer are used in the treatment protocol, theNHE-inhibitor and FAP may be administered together or in a“dual-regimen” wherein the two therapeutics are dosed and administeredseparately. When the NHE inhibitor and the fluid-absorbing polymer aredosed separately, the typical dosage administered to the subject in needof the NHE inhibitor is typically from about 5 mg per day and about 5000mg per day and, in other embodiments, from about 50 mg per day and about1000 mg per day. Such dosages may induce fecal excretion of sodium (andits accompanying anions), from about 10 mmol up to about 250 mmol perday, from about 20 mmol to about 70 mmol per day or even from about 30mmol to about 60 mmol per day.

The typical dose of the fluid-absorbing polymer is a function of theextent of fecal secretion induced by the non-absorbable NHE inhibitor.Typically the dose is adjusted according to the frequency of bowelmovements and consistency of the stools. More specifically the dose isadjusted so as to avoid liquid stools and maintain stool consistency as“soft” or semi-formed, or formed. To achieve the desired stoolconsistency and provide abdominal relief to patients, typical dosageranges of the fluid-absorbing polymer to be administered in combinationwith the NHE inhibitor, are from about 2 g to about 50 g per day, fromabout 5 g to about 25 g per day or even from about 10 g to about 20 gper day. When the NHE-inhibitor and the FAP are administered as a singledosage regimen, the daily uptake may be from about 2 g to about 50 g perday, from about 5 g to about 25 g per day, or from about 10 g to about20 g per day, with a weight ratio of NHE inhibitor to fluid-absorbingpolymer being from about 1:1000 to 1:10 or even from about 1:500 to 1:5or about 1:100 to 1:5.

A typical dosage of the substantially impermeable or substantiallysystemically non-bioavailable. NHE-inhibiting compound when used alonewithout a FAP may be between about 0.2 mg per day and about 2 g per day,or between about 1 mg and about 1 g per day, or between about 5 mg andabout 500 mg, or between about 10 mg and about 250 mg per day, which isadministered to a subject in need of treatment.

The frequency of administration of therapeutics described herein mayvary from once-a-day (QD) to twice-a-day (BID) or thrice-a-day (TID),etc., the precise frequency of administration varying with, for example,the patient's condition, the dosage, etc. For example, in the case of adual-regimen, the NHE-inhibitor could be taken once-a-day while thefluid-absorbing polymer could be taken at each meal (TID).

E. Modes of Administration

The substantially impermeable or substantially systemicallynon-bioavailable, NHE-inhibiting compounds of the present disclosurewith or without the fluid-absorbing polymers described herein may beadministered by any suitable route. The compound is preferablyadministrated orally (e.g., dietary) in capsules, suspensions, tablets,pills, dragees, liquids, gels, syrups, slurries, and the like. Methodsfor encapsulating compositions (such as in a coating of hard gelatin orcyclodextran) are known in the art (Baker, et al., “Controlled Releaseof Biological Active Agents”, John Wiley and Sons. 1986). The compoundscan be administered to the subject in conjunction with an acceptablepharmaceutical carrier as part of a pharmaceutical composition. Theformulation of the pharmaceutical composition will vary according to theroute of administration selected. Suitable pharmaceutical carriers maycontain inert ingredients which do not interact with the compound. Thecarriers are biocompatible, i.e., non-toxic, non-inflammatory,non-immunogenic and devoid of other undesired reactions at theadministration site. Examples of pharmaceutically acceptable carriersinclude, for example, saline, commercially available inert gels, orliquids supplemented with albumin, methyl cellulose or a collagenmatrix. Standard pharmaceutical formulation techniques can be employed,such as those described in Remington's Pharmaceutical Sciences, MackPublishing Company, Easton. Pa.

In other embodiments, the NHE-3 inhibiting compounds may be systemicallyadministered. In one embodiment, the compounds of the present inventionare administered systemically to inhibit NHE-3 in the kidney. Withoutbeing held to any particular theory, the impermeable NHE-inhibitingcompounds (e.g., NHE-3, -2 and/or -8 inhibitors) of the presentdisclosure can also be administered parenterally, by intravenous,subcutaneous or intramuscular injection or infusion to inhibit NHE3 inthe kidney. NHE3 is expressed at high levels on the apical surface ofthe proximal tubule of the kidney and couples luminal Na reabsorption tothe secretion of intracellular protons. Since NHE3 accounts forapproximately 60-80% of sodium reabsorption in the kidney, it isanticipated that NHE inhibition could permit the removal of substantialquantities of systemic fluid and sodium to prevent edema and resolvecongestive heart failure symptoms. This effect could be achieved by NHEinhibition in combination with other diuretics, specifically loopdiuretics, like furosemide, to inhibit tubuloglomerular feedback. Inaddition, since sodium reabsorption via NHE3 in the proximal tubule isresponsible for a large proportion of the energy requirement of theproximal tubule cell, it is anticipated that NHE inhibition in thekidney could be beneficial by reducing the energy requirement andprotecting the proximal tubule cell in settings of decreased energyavailability to the proximal tubule, such as those that occur as aresult of kidney hypoxia such as in kidney ischemia reperfusion injuryresulting in acute kidney injury.

Pharmaceutical preparations for oral use can be obtained by combining acompound of the present disclosure with a solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are, in particular, fillers such assugars, including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of a suitable material, such as gelatin, as well as soft,sealed capsules made of a suitable material, for example, gelatin, and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the activecompounds can be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers can be added. All formulations for oraladministration should be in dosages suitable for such administration.

It will be understood that, certain compounds of the disclosure may beobtained as different stereoisomers (e.g., diastereomers andenantiomers) or as isotopes and that the disclosure includes allisomeric forms, racemic mixtures and isotopes of the disclosed compoundsand a method of treating a subject with both pure isomers and mixturesthereof, including racemic mixtures, as well as isotopes. Stereoisomerscan be separated and isolated using any suitable method, such aschromatography.

F. Delayed Release

NHE proteins show considerable diversity in their patterns of tissueexpression, membrane localization and functional roles. (See, e.g., Thesodium-hydrogen exchanger-From molecule To Its Role In Disease,Karmazyn, M., Avkiran, M., and Fliegel, L., eds., Kluwer Academics(2003).)

In mammals, nine distinct NHE genes (NHE-1 through -9) have beendescribed. Of these nine, five (NHE-1 through -5) are principally activeat the plasma membrane, whereas NHE-6, -7 and -9 reside predominantlywithin intracellular compartments.

NHE-1 is ubiquitously expressed and is chiefly responsible forrestoration of steady state intracellular pH following cytosolicacidification and for maintenance of cell volume. Recent findings showthat NHE-1 is crucial for organ function and survival (e.g. NHE-1-nullmice exhibit locomotor abnormalities, epileptic-like seizures andconsiderable mortality before weaning).

In contrast with NHE-1 expressed at the basolateral side of the nephronsand gut epithelial cells, NHE-2 through -4 are predominantly expressedon the apical side of epithelia of the kidney and the gastrointestinaltract. Several lines of evidence show that NHE-3 is the majorcontributor of renal bulk Na+ and fluid re-absorption by the proximaltubule. The associated secretion of H+ by NHE-3 into the lumen of renaltubules is also essential for about ⅔ of renal HCO₃ ⁻ re-absorption.Complete disruption of NHE-3 function in mice causes a sharp reductionin HCO₃ ⁻, Na+ and fluid re-absorption in the kidney, which isconsistently associated with hypovolemia and acidosis.

In one embodiment, the novel compounds of the invention are intended totarget the apical NHE antiporters (e.g. NHE-3, NHE-2 and NHE-8) withoutsubstantial permeability across the layer of gut epithelial cells,and/or without substantial activity towards NHEs that do not residepredominantly in the GI tract. This invention provides a method toselectively inhibit GI apical NHE antiporters and provide the desiredeffect of salt and fluid absorption inhibition to correct abnormal fluidhomeostasis leading to constipations states. Because of their absence ofsystemic exposure, said compounds do not interfere with other keyphysiological roles of NHEs highlighted above. For instance, thecompounds of the invention are expected to treat constipation inpatients in need thereof, without eliciting undesired systemic effects,such as for example salt wasting or bicarbonate loss leading tohyponatriemia and acidosis among other disorders.

In another embodiment, the compounds of the invention are delivered tothe small bowel with little or no interaction with the upper GI such asthe gastric compartment and the duodenum. The applicant found that anearly release of the compounds in the stomach or the duodenum can havean untoward effect on gastric secretion or bicarbonate secretion (alsoreferred to as “bicarbonate dump”). In this embodiment the compounds aredesigned so as to be released in an active form past the duodenum. Thiscan be accomplished by either a prodrug approach or by specific drugdelivery systems.

As used herein, “prodrug” is to be understood to refer to a modifiedform of the compounds detailed herein that is inactive (or significantlyless active) in the upper GI, but once administered is metabolised invivo into an active metabolite after getting past, for example, theduodenum. Thus, in a prodrug approach, the activity of the NHE inhibitorcan be masked with a transient protecting group that is liberated aftercompound passage through the desired gastric compartment. For example,acylation or alkylation of the essential guanidinyl functionality of theNHE inhibitor would render it biochemically inactive; however, cleavageof these functional groups by intestinal amidases, esterases,phosphatases, and the like, as well enzymes present in the colonicflora, would liberate the active parent compound. Prodrugs can bedesigned to exploit the relative expression and localization of suchphase I metabolic enzymes by carefully optim/zing the structure of theprodrug for recognition by specific enzymes. As an example, theanti-inflammatory agent sulfasalazine is converted to 5-aminosalicylatein the colon by reduction of the diazo bond by intestinal bacteria.

In a drug delivery approach the NHE-inhibitor compounds of the inventionare formulated in certain pharmaceutical compositions for oraladministration that release the active in the targeted areas of the GI,i.e., jejunum, ileum or colon, or preferably the distal ileum and colon,or even more preferably the colon.

Methods known from the skilled-in-the-art are applicable. (See, e.g.,Kumar, P. and Mishra. B., Colon Targeted Drug Delivery Systems—AnOverview. Curr. Drug Deliv., 2008, 5 (3), 186-198; Jain, S. K. and Jain,A., Target-specific Drug Release to the Colon, Epert Opin. Drug Deliv.,2008, 5 (5), 483-498: Yang, L., Biorelevant Dissolution Testing ofColon-Specific Delivery Systems Activated by Colonic Microflora, J.Control Release, 2008, 125 (2), 77-86; Siepmann, F.; Siepmann, J.;Walther, M.; MacRae, R. J.; and Bodmeier, R., Polymer Blends forControlled Release Coatings, J. Control Release 2008, 125 (1), 1-15;Patel, M.; Shah, T.; and Amin, A., Therapeutic Opportunities inColon-Specific Drug-Delivery Systems, Crit. Rev. Ther. Drug CarrierSyst., 2007, 24 (2), 147-202; Jain, A.; Gupta, Y.; Jain, S. K.,Perspectives of Biodegradable Natural Polysaccharides for Site-specificDrug Delivery to the Colon, J. Pharm. Sci., 2007, 10 (1), 86-128: Vanden, M. G., Colon Drug Delivery, Expert Opin. Drug Deliv., 2006, 3 (1),111-125; Basit, A. W., Advances in Colonic Drug Delivery, Drugs 2005, 65(14), 1991-2007; Chourasia, M. K.; Jain, S. K., Polysaccharides forColon-Targeted Drug Delivery, Drug Deliv. 2004, 11 (2), 129-148;Shareef, M. A.; Khar, R. K.; Ahuja. A.; Ahmad, F. J.; and Raghava, S.,Colonic Drug Delivery: An Updated Review, AAPS Pharm. Sci. 2003, 5 (2).E17: Chourasia, M. K.; Jain, S. K., Pharmaceutical Approaches to ColonTargeted Drug Delivery Systems, J. Pharm. Sci. 2003, 6 (1), 33-66; and,Sinha, V. R.; Kumria, R., Colonic Drug Delivery; Prodrug Approach,Pharm. Res. 2001, 18 (5), 557-564. Typically the active pharmaceuticalingredient (API) is contained in a tablet/capsule designed to releasesaid API as a function of the environment (e.g., pH, enzymatic activity,temperature, etc.), or as a function of time. One example of thisapproach is Eudracol™ (Pharma Polymers Business Line of Degussa'sSpecialty Acrylics Business Unit), where the API-containing core tabletis layered with various polymeric coatings with specific dissolutionprofiles. The first layer ensures that the tablet passes through thestomach intact so it can continue through the small intestine. Thechange from an acidic environment in the stomach to an alkalineenvironment in the small intestine initiates the release of theprotective outer layer. As it travels through the colon, the next layeris made permeable by the alkalinity and intestinal fluid. This allowsfluid to penetrate to the interior layer and release the activeingredient, which diffuses from the core to the outside, where it can beabsorbed by the intestinal wall. Other methods are contemplated withoutdeparting from the scope of the present disclosure.

In another example, the pharmaceutical compositions of the invention canbe used with drug carriers including pectin and galactomannan,polysaccharides that are both degradable by colonic bacterial enzymes.(See, e.g., U.S. Pat. No. 6,413,494, the entire contents of which areincorporated herein by reference for all relevant and consistentpurposes.) While pectin or galactomannan, if used alone as a drugcarrier, are easily dissolved in simulated gastric fluid and simulatedintestinal fluid, a mixture of these two polysaccharides prepared at apH of about 7 or above produces a strong, elastic, and insoluble gelthat is not dissolved or disintegrated in the simulated gastric andintestinal fluids, thus protecting drugs coated with the mixture frombeing released in the upper GI tract. When the mixture of pectin andgalactomannan arrives in the colon, it is rapidly degraded by thesynergic action of colonic bacterial enzymes. In yet another aspect, thecompositions of the invention may be used with the pharmaceutical matrixof a complex of gelatin and an anionic polysaccharide (e.g., pectinate,pectate, alginate, chondroitin sulfate, polygalacturonic acid,tragacanth gum, arabic gum, and a mixture thereof), which is degradableby colonic enzymes (U.S. Pat. No. 6,319,518).

In yet other embodiments, fluid-absorbing polymers that are administeredin accordance with treatment methods of the present disclosure areformulated to provide acceptable/pleasant organoleptic properties suchas mouthfeel, taste, and/or to avoid premature swelling/gelation in themouth and in the esophagus and provoke choking or obstruction. Theformulation may be designed in such a way so as to ensure the fullhydration and swelling of the FAP in the GI tract and avoid theformation of lumps. The oral dosages for the FAP may take various formsincluding, for example, powder, granulates, tablets, wafer, cookie andthe like, and are most preferably delivered to the small bowel withlittle or no interaction with the upper GI such as the gastriccompartment and the duodenum.

The above-described approaches or methods are only some of the manymethods reported to selectively deliver an active in the lower part ofthe intestine, and therefore should not be viewed to restrain or limitthe scope of the disclosure.

The following non-limiting examples are provided to further illustratethe present disclosure.

EXAMPLES Exemplary Compound Synthesis Example 12-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethylphosphonicacid

Intermediate 1.1: 2-bromo-1-(3-bromophenyl)ethanone

Into a 500-mL 3-necked round-bottom flask, was placed a solution of1-(3-bromophenyl)ethanone (40 g, 202.02 mmol, 1.00 equiv) in acetic acid(200 mL). This was followed by the addition of a solution of Br₂ (32 g,200.00 mmol) in acetic acid (50 mL) dropwise with stirring at 60° C. Theresulting solution was stirred for 3 h at 60° C. in an oil bath. Theresulting mixture was concentrated under vacuum. The crude product wasre-crystallized from petroleum ether:ethyl acetate in the ratio of 8:1.This resulted in 24 g (43%) of 2-bromo-1-(3-bromophenyl)ethanone as ayellow solid.

Intermediate 1.2:1-(3-bromophenyl)-2-((2,4-dichlorobenzyl)(methyl)amino)ethanone

Into a 1 L 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of2-bromo-1-(3-bromophenyl)ethanone (55 g, 199.28 mmol, 1.00 equiv) in1,4-dioxane (300 mL), TEA (40 g, 396.04 mmol, 1.99 equiv), and(2,4-dichlorophenyl)-N-methylmethanamine (38 g, 201.06 mmol, 1.01equiv). The resulting solution was stirred for 2 h at 25° C. in an oilbath. The solids were filtered out and the filtrate was used without anyfurther purification.

Intermediate 1.3:1-(3-bromophenyl)-2-((2,4-dichlorobenzyl)(methyl)amino)ethanol

Into a 1 L 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of2-((2,4-dichlorobenzyl)(methyl)amino)-1-(3-bromophenyl)ethanone (77 g,198.97 mmol, 1.00 equiv, theoretical yield) in methanol (300 mL). Thiswas followed by the addition of NaBH₄ (15 g, 394.74 mmol, 1.98 equiv) inseveral batches at 0° C. The resulting solution was stirred for 30 minat 0° C. in a water/ice bath. The reaction was then quenched by theaddition of 100 mL of acetone. The resulting mixture was concentratedunder vacuum. The resulting solution was extracted with 3×100 mL ofethyl acetate and the organic layers combined and dried over anhydroussodium sulfate. The residue was applied onto a silica gel column withethyl acetate:petroleum ether (1:100). This resulted in 50 g (65%) of2-((2,4-dichlorobenzyl)(methyl)amino)-1-(3-bromophenyl)ethanol as ayellow oil.

Intermediate 1.4:4-(3-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline

Into a 500-mL 3-necked round-bottom flask, was placed a solution of2-((2,4-dichlorobenzyl)(methyl)amino)-1-(3-bromophenyl)ethanol (25 g,64.27 mmol, 1.00 equiv) in dichloromethane (100 mL). This was followedby the addition of sulfuric acid (100 mL) dropwise with stirring at 0-5°C. The resulting solution was stirred for 4 h at room temperature. Theresulting solution was diluted with of ice water. The pH value of thesolution was adjusted to 8 with sodium hydroxide. The resulting solutionwas extracted with 3×300 mL of dichloromethane and the organic layerscombined and dried over anhydrous sodium sulfate and concentrated undervacuum. The crude product was re-crystallized from petroleum ether:ethylacetate in the ratio of 8:1. This resulted in 15 g (63%) of4-(3-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolineas a white solid.

Intermediate 1.5:4-(3-(benzylthio)phenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of potassiumcarbonate (930 mg, 0.50 equiv) in xylene (50 mL). This was followed bythe addition of phenylmethanethiol (2.5 g, 1.50 equiv) dropwise withstirring at 0° C. The resulting solution was stirred for 1 h at 25° C.Into another 100-mL 3-necked round-bottom flask purged and maintainedwith an inert atmosphere of nitrogen, was added a solution of4-(3-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline(5.0 g, 1 equiv) in xylene (50 mL), Pd₂(dba)₃ (300 mg), Xantphos (300mg). The resulting solution was stirred for 30 min at 25° C. and thenadded to the above reaction solution. The mixture was stirred overnightat 140° C. The resulting mixture was concentrated under vacuum. Theresidue was applied onto a silica gel column with ethylacetate/petroleum ether (1:100˜1:50). This resulted in 2.5 g (45%) of4-(3-(benzylthio)phenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolineas a yellow oil.

Intermediate 1.6:3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride

Into a 250-mL 3-necked round-bottom flask, was placed a solution of4-(3-(benzylthio)phenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline(8 g, 13.53 mmol, 1.00 equiv, 70%) in acetic acid/water (80/8 mL).Cl₂(g) was introduced and the resulting solution was stirred for 1 h atroom temperature. The resulting mixture was concentrated under vacuum.This resulted in 5.0 g (90%) of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride hydrochloride as a yellowish solid.

Intermediate 1.7: 2-(2-bromoethyl)isoindoline-1,3-dione

Into a 500-mL round-bottom flask, was placed a solution of1,2-dibromoethane (30 g, 159.57 mmol, 2.95 equiv) inN,N-dimethylformamide (200 mL). This was followed by the addition ofpotassium phthalimide (10 g, 54.05 mmol, 1.00 equiv) in several batches.The resulting solution was stirred for 24 h at 60° C. The reaction wasthen quenched by the addition of 500 mL of water. The resulting solutionwas extracted with 2×200 mL of ethyl acetate and the organic layerscombined and dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was applied onto a silica gel column with ethylacetate/petroleum ether (1:10). This resulted in 8 g (57%) of2-(2-bromoethyl)isoindoline-1,3-dione as a white solid.

Intermediate 1.8: diethyl 2-(1,3-dioxoisoindolin-2-yl)ethylphosphonate

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed 2-(2-bromoethyl)isoindoline-13-dione(8 g, 31.50 mmol, 1.00 equiv) and triethyl phosphite (6.2 g, 37.35 mmol,1.19 equiv). The resulting solution was stirred for 18 h at 130° C. Theresulting mixture was concentrated under vacuum. The crude product wasre-crystallized from ether:n-hexane (1:2). This resulted in 5 g (48%) ofdiethyl 2-(1,3-dioxoisoindolin-2-yl)ethylphosphonate as a white solid.

Intermediate 1.9: diethyl 2-aminoethylphosphonate

Into a 500-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of diethyl2-(1,3-dioxoisoindolin-2-yl)ethylphosphonate (5 g, 16.08 mmol, 1.00equiv) in ethanol (200 mL) and hydrazine hydrate (8 g, 160.00 mmol, 9.95equiv). The resulting solution was stirred for 12 h at room temperature.The solids were filtered and the resulting mixture was concentratedunder vacuum. The residue was applied onto a silica gel column andeluted with dichloromethane/methanol (9:1). This resulted in 1.5 g (51%)of diethyl 2-aminoethylphosphonate as colorless oil.

Intermediate 1.10: Diethyl2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethylphosphonate

Into a 50-mL round-bottom flask, was placed a solution of diethyl2-aminoethylphosphonate (100 mg, 0.55 mmol, 1.00 equiv) indichloromethane (10 mL) with TEA (220 mg, 2.18 mmol, 3.94 equiv). Thiswas followed by the addition of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (300 mg, 0.60 mmol, 1.08 equiv, 78%) in several batches. Theresulting solution was stirred for 2 h at room temperature. The reactionprogress was monitored by LCMS. The resulting mixture was concentratedunder vacuum. The residue was applied onto a silica gel column withdichloromethane:methanol (50:1). This resulted in 0.07 g (24%) of thetitle compound as a colorless oil.

Compound 1:2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethylphosphonicacid

To a solution of Intermediate 1.10 (70 mg, 0.13 mmol, 1.00 equiv) indichloromethane (10 mL) was added bromotrimethylsilane (200 mg, 1.32mmol, 10.04 equiv). The resulting solution was stirred overnight at 40°C. in an oil bath. The reaction progress was monitored by LCMS. Theresulting mixture was concentrated under vacuum. To the above was addedmethanol. The resulting mixture was concentrated under vacuum. This wasfollowed by the addition of a solution of sodium hydroxide (11 mg, 0.28mmol, 2.10 equiv) in methanol (2 mL). The resulting solution was stirredfor an additional 1 h at room temperature. The resulting mixture wasconcentrated under vacuum. The solid was dried in an oven under reducedpressure. This resulted in 52.3 mg (73%) of the title compound as asodium salt. ¹H-NMR (300 MHz, CD₃OD, ppm): 7.82 (d, J=7.5 Hz, 1H), 7.73(s, 1H), 7.56 (m, 1H), 7.48 (d, J=8.1 Hz, 1H), 7.41 (s, 1H), 6.88 (s,1H), 4.54 (s, 1H), 3.97 (m, 2H), 3.17 (m, 3H), 2.97 (m, 1H), 2.67 (s,3H), 1.68 (m, 2H). MS (ES, m/z): 479 [M+H]⁺.

Example 24-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)phenylphosphonicacid

Intermediate 2.1: diethyl 4-nitrophenylphosphonate

Into a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of diethylphosphonate (3.02 g, 21.88 mmol, 1.10 equiv) in toluene (10 mL),Pd(PPh₃)₄ (1.15 g, 1.00 mmol, 0.05 equiv), TEA (2.21 g, 21.88 mmol, 1.10equiv), 1-bromo-4-nitrobenzene (4 g, 19.90 mmol, 1.00) equiv). Theresulting solution was stirred for 15 h at 90° C. The solids werefiltered out and the resulting mixture was concentrated under vacuum.The residue was applied onto a silica gel column and eluted with ethylacetate/petroleum ether (1:2). This resulted in 3.53 g (68%) of diethyl4-nitrophenylphosphonate as a yellow liquid.

Intermediate 2.2: diethyl 4-aminophenylphosphonate

Into a 50-mL round-bottom flask, was placed a solution of diethyl4-nitrophenylphosphonate (1.07 g, 4.13 mmol, 1.00 equiv), TEA (3 mL),Palladium carbon (0.025 g). This was followed by the addition of formicacid (2 mL) dropwise with stirring at room temperature. The resultingsolution was heated to reflux for 3 hr. The reaction was then quenchedby the addition of 5 mL of water and the solids were filtered out. Theresulting filtrate was extracted with 5×10 mL of dichloromethane and theorganic layers combined and dried over anhydrous sodium sulfate. Thisresulted in 800 mg (85%) of diethyl 4-aminophenylphosphonate as a whitesolid.

Compound 2:4-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl-sulfonamido)phenylphosphonicacid

Compound 2 was prepared in an analogous manner to that of Compound 1using diethyl 4-aminophenylphosphonate (Intermediate 2.2) as the amine.¹H-NMR (300 MHz, CD₃OD, ppm): 7.86 (d, 1H), 7.69 (m, 3H), 7.55 (m, 3H),7.21 (m, 2H), 6.73 (s, 1H), 4.70 (m, 2H), 4.48 (d, 1H), 3.79 (m, 1H),3.46 (m, 1H), 3.09 (s, 3H). MS (ES, m/z): 527 [M+H]+.

Example 34-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)benzylphosphonicacid

Intermediate 3.1: diethyl 4-nitrobenzylphosphonate

Into a 250-mL round-bottom flask, was placed1-(bromomethyl)-4-nitrobenzene (15 g, 69.77 mmol, 1.00 equiv), triethylphosphite (70 mL). The resulting solution was stirred for 2 h at 110° C.in an oil bath. The resulting mixture was concentrated under vacuum. Theresidue was applied onto a silica gel column with ethylacetate/petroleum ether (1:10-1:1). This resulted in 17 g (89%) of thetitle compound as a yellow oil.

Intermediate 3.2: diethyl 4-aminobenzylphosphonate

Into a 100-mL 3-necked round-bottom flask, was placed a solution ofdiethyl 4-nitrobenzylphosphonate (5 g, 18.32 mmol, 1.00 equiv) inethanol (50 mL) and a solution of NH₄Cl (2.9 g, 54.72 mmol, 2.99 equiv)in water (50 mL) was added. This was followed by the addition of Fe (4.1g, 73.21 mmol, 4.00 equiv), while the temperature was maintained atreflux. The resulting solution was heated to reflux for 1 hr. The solidswere filtered out. The resulting mixture was concentrated under vacuum.The resulting solution was extracted with 3×20 mL of ethyl acetate andthe organic layers combined and dried over anhydrous sodium sulfate. Thesolids were filtered out. The resulting mixture was concentrated undervacuum. The residue was applied onto a silica gel column and eluted withethyl acetate/petroleum ether (1:3). This resulted in 2.5 g (56%) of thetitle compound as a yellow solid.

Compound 3:4-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)benzylphosphonicacid

Compound 3 was prepared in an analogous manner to that of Compound 1using diethyl 4-aminobenzylphosphonate (Intermediate 3.2) as the amine.¹H-NMR (300 MHz, CD₃OD, ppm): 7.89 (d, J=7.8 Hz, 1H), 7.61˜7.66 (m, 1H),7.52˜7.54 (m, 2H), 7.21˜7.20 (m, 2H), 7.11 (s, 1H), 6.95 (d, J=8.1 Hz,2H), 6.73 (s, 1H), 4.51˜4.59 (m, 3H), 3.33 (s, 1H), 3.03˜2.89 (m, 6H).MS (ES, m/z): 541 [M+H]⁺.

Example 43-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)propylphosphonicacid

Intermediate 4.1: 3-diethyl 3-aminopropylphosphonate

Following the procedures outlined in Example 1, substitutingdibromopropane for dibromoethane gave the title compound.

Compound 43-(3-(6,8-dichloro-2-methyl-1,2,34-tetrahydroisoquinolin-4-yl)phenylsulfonamido)propylphosphonicacid

Compound 4 was prepared in an analogous manner to that of Compound 1using 3-diethyl 3-aminopropylphosphonate (Intermediate 4.1) as theamine. ¹H-NMR (300 MHz, CD₃OD, ppm): 7.87 (d, J=8.1 Hz, 1H), 7.77 (s,1H), 7.61˜7.66 (m, 1H), 7.51˜7.54 (m, 2H), 6.88 (s, 1H), 4.77˜4.83 (m,1H), 4.65 (d, J=16.2 Hz, 1H), 4.44 (d, J=15.6 Hz, 1H), 3.78˜3.84 (m,1H), 3.50˜3.57 (m, 1H), 3.08 (s, 3H), 2.93˜2.97 (m, 2H), 1.61˜1.72 (m,2H), 1.48˜1.59 (m, 2H). MS (ES, m/z): 493 [M+H]⁺.

Example 5(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)methylphosphonicacid

Intermediate 5.1: 1,3,5-tribenzyl-1,3,5-triazinane

Into a 100-mL 3-necked round-bottom flask was placed benzylamine (10 g,93.46 mmol, 1.00 equiv), followed by the addition of formaldehyde (9.0g, 1.20 equiv, 37%) dropwise with stirring at 0-10° C. To theprecipitated gum was added 3M aqueous sodium hydroxide (20 mL), and themixture was stirred. After standing in ice for 0.3 h, ether (30 mL) wasadded, and the mixture stirred until all precipitate dissolved. Theaqueous phase was separated and extracted with ether. The solvents wereremoved under vacuum to afford 12 g (36%) of1,3,5-tribenzyl-1,3,5-triazinane as colorless oil.

Intermediate 5.2: diethyl (benzylamino)methylphosphonate

Into a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed1,3,5-tribenzyl-1,3,5-triazinane (3.0 g, 8.40 mmol, 1.00 equiv) anddiethyl phosphite (3.5 g, 25.36 mmol, 3.00 equiv). The resultingsolution was stirred for 3 h at 100° C. The residue was applied onto asilica gel column with ethyl acetate/petroleum ether (1:20 to 1:1). Thisresulted in 2.0 g (90%) of diethyl (benzylamino)methylphosphonate as acolorless oil.

Intermediate 5.3: Diethyl aminomethylphosphonate

A 250-mL pressure tank reactor was purged, flushed and maintained with ahydrogen atmosphere, then, was added a solution of diethyl(benzylamino)methylphosphonate (3.5 g, 13.62 mmol, 1.00 equiv) inethanol (180 mL), acetic acid (10 mL) and Palladium carbon (0.2 g, 0.10equiv). The resulting solution was stirred for 24 h at 50° C. under 20atm pressure. The solids were filtered out. The resulting mixture wasconcentrated under vacuum. This resulted in 2.0 g (crude) of the titlecompound as brown oil which was used without further purification.

Compound 5:(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)methylphosphonicacid

Compound 5 was prepared in an analogous manner to that of Compound 1using diethyl aminomethylphosphonate (Intermediate 5.3) as the amine.¹H-NMR (300 MHz, CD₃OD, ppm): 7.89 (d, J=7.8 Hz, 1H), 7.74 (s, 1H),7.63˜7.66 (m, 1H), 7.57˜7.61 (m, 2H), 6.97 (s, 1H), 4.80˜4.89 (m, 1H),4.55˜4.67 (m, 2H), 3.83˜3.89 (m, 1H), 3.55˜3.66 (m, 1H), 3.02˜3.11 (m,5H). MS (ES, m/z): 465 [M+H]⁺.

Example 64-((3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)methyl)benzylphosphonicacid

Intermediate 6.1: 4-diethyl 4-(aminomethyl)benzylphosphonate

Following the procedures outlined in Example 1, substituting1,4-bis(bromomethyl)benzene for dibromoethane gave the title compound.

Compound 64-((3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)methyl)benzylphosphonicacid

Compound 6 was prepared in an analogous manner to that of Compound 1using 4-diethyl 4-(aminomethyl)benzylphosphonate (Intermediate 6.1) asthe amine. ¹H-NMR (300 MHz, CD₃OD, ppm): 7.85˜7.88 (m, 1H), 7.54˜7.59(m, 2H), 7.37˜7.42 (m, 2H), 7.198˜7.22 (m, 2H), 7.06˜7.09 (m, 1H), 6.77(s, 1H), 4.64 (m, J=16.2 Hz, 1H), 4.49˜4.53 (m, 1H), 4.37 (m, J=16.5,1H), 4.17 (s, 2H), 3.45˜3.56 (m, 1H), 3.11˜3.27 (m, 1H), 3.09˜3.10 (m,4H), 2.96˜2.97 (m, 1H). MS (ES, m/z): 555 [M+H]⁺.

Example 73-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)propane-1-sulfonicacid

Compound 7:3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)propane-1-sulfonicacid

Into a 50-mL round-bottom flask, was placed a solution of3-aminopropane-1-sulfonic acid (180 mg, 1.29 mmol, 1.00 equiv) intetrahydrofuran/water (10/10 mL) with sodium bicarbonate (430 mg, 5.12mmol). This was followed by the addition of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (500 mg, 1.29 mmol, 0.99 equiv) in several batches. Theresulting solution was stirred for 4 h at room temperature. The reactionprogress was monitored by LCMS. The pH value of the solution wasadjusted to 6 with 1M hydrogen chloride. The resulting mixture wasconcentrated under vacuum. The crude product (500 mg) was purified bypreparative HPLC to give 26.7 mg of the title compound (4%) as a TFAsalt. ¹H-NMR (300 MHz, DMSO, ppm): 10.28 (s, 1H), 7.53˜7.79 (m, 6H),6.83 (s, 1H), 4.74 (s, 2H), 4.51 (s, 1H), 3.90 (s, 1H), 3.06 (s, 3H),2.86˜2.93 (m, 2H), 2.33˜2.44 (m, 2H), 1.58˜1.63 (m, 2H). MS (ES, m/z):493 [M+H]⁺.

Example 82-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)-N-(phosphonomethyl)phenylsulfonamido)aceticacid

Intermediate 8.1: ethyl2-(benzyl((diethoxyphosphoryl)methyl)amino)acetate

Into a 500-mL 3-necked round-bottom flask, was placed a solution ofdiethyl (benzylamino)methylphosphonate (intermediate 5.2) (12 g, 46.69mmol, 1.00 equiv) in acetonitrile (150 mL). DIEA (12 g, 2.00 equiv).This was followed by the addition of ethyl 2-bromoacetate (8.4 g, 50.30mmol, 1.10 equiv) dropwise with stirring. The mixture was stirred for 30min at room temperature. The resulting solution was heated to reflux for6 hr. The resulting mixture was cooled to room temperature andconcentrated under vacuum. The residue was applied onto a silica gelcolumn with ethyl acetate/petroleum ether (1:20 to 1:5). This resultedin 8.0 g (50%) of ethyl2-(benzyl((diethoxyphosphoryl)methyl)amino)acetate as yellow oil.

Intermediate 8.2: ethyl 2-((diethoxyphosphoryl)methylamino)acetate

A 250-mL pressure tank reactor was purged, flushed and maintained with ahydrogen atmosphere, then, was added a solution of ethyl2-(benzyl((diethoxyphosphoryl)methyl)amino)acetate (8.0 g, 23.32 mmol,1.00 equiv) in ethanol (180 mL), acetic acid (10 mL), Pd/C (0.9 g). Theresulting solution was stirred at 20 atm for 32 h at 50° C. The solidswere filtered out, and the resulting mixture was concentrated undervacuum. This resulted in 6.0 g (82%) of the acetic acid salt of ethyl2-((diethoxyphosphoryl)methylamino)acetate as a brown oil.

Intermediate 8.3: ethyl2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)-N-((diethoxyphosphoryl)methyl)phenylsulfonamido)acetate

Into a 50-mL round-bottom flask, was placed a solution of ethyl2-((diethoxyphosphoryl)methylamino)acetate (320 mg, 1.26 mmol, 1.00equiv) in pyridine (10 mL),3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (500 mg, 1.28 mmol, 1.01 equiv) was added and the resultingsolution was stirred overnight at room temperature. The reactionprogress was monitored by LCMS. The resulting mixture was concentratedunder vacuum. The crude product (400 mg) was purified by preparativeHPLC to give 200 mg (24%) of the title compound as a TFA salt.

Intermediate 8.4:(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)-N-(2-ethoxy-2-oxoethyl)phenylsulfonamido)methylphosphonicacid

Into a 50-mL round-bottom flask, was placed a solution of Intermediate8.3 (200 mg, 0.33 mmol, 1.00 equiv) in dichloromethane (6 mL).Bromotrimethylsilane (502 mg, 3.30 mmol, 10.01 equiv) was added and theresulting solution was stirred overnight at 40° C. in an oil bath. Thereaction progress was monitored by LCMS. The resulting mixture wasconcentrated under vacuum. The residue was dissolved in 10 mL ofmethanol. The resulting mixture was concentrated under vacuum. Thisresulted in 180 mg (9%) of the title compound as a yellow solid.

Compound 8:2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)-N-(phosphonomethyl)phenylsulfonamido)aceticacid

Into a 50-mL round-bottom flask, was placed a solution of(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)-N-(2-ethoxy-2-oxoethyl)phenylsulfonamido)methylphosphonicacid (Intermediate 8.4) (180 mg, 0.33 mmol, 1.00 equiv) intetrahydrofuran/water (5/5 mL). This was followed by the addition oflithium hydroxide (39 mg, 1.62 mmol, 4.97 equiv) in several batches atroom temperature. The resulting solution was stirred for 4 h at roomtemperature. The reaction progress was monitored by LCMS. The resultingmixture was concentrated under vacuum. The pH value of the solution wasadjusted to 6 with 1M hydrogen chloride. The resulting mixture wasconcentrated under vacuum. The crude product (150 mg) was purified bypreparative HPLC giving 59.2 mg (35%) of the title compound as a TFAsalt. ¹H˜NMR (300 MHz, DMSO+D₂O, ppm): 7.73˜7.74 (m, 1H), 7.67˜7.68 (m,1H), 7.58˜7.62 (m, 2H), 7.49 (s, 1H), 7.00 (s, 1H), 4.71˜4.75 (m, 1H),4.49 (d, J=16.2 Hz, 1H), 4.33 (d, J=15.9 Hz, 1H), 4.07 (s, 2H),3.62˜3.64 (m, 1H), 3.45˜3.54 (m, 2H), 3.31˜3.40 (m, 1H), 2.88 (s, 3H).MS (ES, m/z): 523 [M+H]⁺.

Example 92-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)succinicacid

Intermediate 9.1: Dimethyl 2-aminosuccinate hydrochloride

Into a 100-mL round-bottom flask, was placed a solution of2-aminosuccinic acid (3 g, 22.56 mmol, 1.00 equiv) in methanol (20 mL).This was followed by the addition of thionyl chloride (10 g, 84.75 mmol,3.76 equiv) dropwise with stirring at 0-5° C. The resulting solution washeated to reflux for 2 h in an oil bath. The resulting mixture wasconcentrated under vacuum. This resulted in 4.2 g (95%) of the titlecompound as a white solid.

Intermediate 9.2: Dimethyl2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)succinate

Into a 50-mL round-bottom flask, was placed a solution of dimethyl2-aminosuccinate hydrochloride (107 mg, 0.54 mmol, 1.00 equiv) inpyridine (5 mL). This was followed by the addition of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (300 mg, 0.69 mmol, 1.27 equiv, 90%) in several batches. Theresulting solution was stirred overnight at room temperature. Theresulting mixture was concentrated under vacuum. The residue was appliedonto a silica gel column with dichloromethane:methanol (50:1). Thisresulted in 200 mg (72%) of the title compound as a colorless oil

Compound 9:2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)succinicacid

Into a 50-mL round-bottom flask, was placed a solution of Intermediate9.2 (100 mg, 0.19 mmol, 1.00 equiv) in tetrahydrofuran (5 mL) and water(5 mL). This was followed by the addition of LiOH (23 mg, 0.96 mmol,4.93 equiv) in several batches at room temperature. The resultingsolution was stirred for 2 h at room temperature. The reaction progresswas monitored by LCMS. The resulting mixture was concentrated undervacuum. The pH value of the solution was adjusted to 6 with hydrogenchloride (1 mol/L). The solids were collected by filtration. The crudeproduct (200 mg) was purified by preparative HPLC to give 12.1 mg (10%)the title compound as a TFA salt. ¹H-NMR (300 MHz, CD₃OD, ppm): 7.89 (d,J=7.2 Hz, 1H), 7.80 (d, J=6.3 Hz, 1H), 7.64˜7.52 (m, 3H), 6.95 (s, 1H),4.78˜4.70 (m, 2H), 4.55˜4.50 (m, 1H), 4.23˜4.17 (m, 1H), 3.87˜3.82 (m,1H), 3.63˜3.57 (m, 1H), 3.12 (s, 3H), 2.79˜2.65 (m, 2H). MS (ES, m/z):487 [M-CF₃COOH+H]⁺.

Example 102-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethylphosphonicacid

Intermediate 10.1: 2-bromo-1-(4-bromophenyl)ethanone

Into a 250-mL 3-necked round-bottom flask, was placed a solution of1-(4-bromophenyl)ethanone (10.0 g, 50.25 mmol, 1.00 equiv) in aceticacid (50 mL). This was followed by the addition of a solution of bromine(8.2 g, 1.05 equiv) in acetic acid (50 mL) dropwise with stirring at 60°C. over 90 min. The resulting solution was stirred for 3 h at 60° C. Theresulting mixture was concentrated under vacuum. The crude product wasre-crystallized from petroleum ether/ethyl acetate in the ratio of 7:1.This resulted in 9.3 g (67%) of the title compound as a yellow solid.

Intermediate 10.2:1-(4-bromophenyl)-2-((2,4-dichlorobenzyl)(methyl)amino)ethanone

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of2-bromo-1-(4-bromophenyl)ethanone (9.3 g, 33.45 mmol, 1.00 equiv) indioxane (100 mL), triethylamine (5.0 g, 1.50 equiv), and(2,4-dichlorophenyl)-N-methylmethanamine (6.4 g, 33.68 mmol, 1.00equiv). The resulting solution was stirred for 2 h at 25° C. The solidswere filtered out. The filtrate was used for next step directly.

Intermediate 10.3:2-((2,4-dichlorobenzyl)(methyl)amino)-1-(4-bromophenyl)ethanol

Into a 500-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of the crudeIntermediate 10.2 in fresh methanol (100 mL). This was followed by theaddition of sodium borohydride (2.5 g, 65.79 mmol, 2.00 equiv) inseveral batches at 0-5° C. The resulting solution was stirred for 1 h at25° C. The reaction was then quenched by the addition of sat. NH₄Cl. Theresulting mixture was concentrated under vacuum. The resulting solutionwas extracted with EtOAc (2×100 mL) and the organic layers combined andconcentrated under vacuum. The crude product was re-crystallized frompetroleum ether/ethyl acetate (60 mL) in the ratio of 7:1. This resultedin 6.5 g (50%) of the title compound as a white solid. MS (ES, m/z): 390[M+H].

Intermediate 10.4:4-(4-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline

Into a 50-mL 3-necked round-bottom flask, was placed a solution of2-((2,4-dichlorobenzyl)(methyl)amino)-1-(4-bromophenyl)ethanol (1.0 g,2.57 mmol, 1.00 equiv) in dichloromethane (3 mL). This was followed bythe addition of conc.H₂SO₄ (2 mL) dropwise with stirring at 0-5° C. Theresulting solution was stirred for 3 h at 20° C. The reaction was thenquenched by the addition of water/ice. The pH value of the solution wasadjusted to 9 with sodium hydroxide. The resulting solution wasextracted with dichloromethane (2×30 mL) and the organic layers combinedand dried over anhydrous sodium sulfate and concentrated under vacuum.This resulted in 0.9 g of the title compound which was used withoutfurther purification. MS (ES, m/z): 372 [M+H]⁺.

Intermediate 10.5:4-(4-(benzylthio)phenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed K₂CO₃ (800 mg, 0.50 equiv) andxylene (50 mL). This was followed by the addition of phenylmethanethiol(1.75 g, 1.00 equiv) dropwise with stirring at 0° C. The resultingmixture was then allowed to warm to room temperature and stirred for 1h. Into another 250-mL 3-necked round-bottom flask purged and maintainedwith an inert atmosphere of nitrogen, was placed4-(4-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline(4.8 g, 0.80 equiv), Xantphos (200 mg, 0.08 equiv) and Pd₂(dba)₃ (200mg, 0.08 equiv) in xylene (30 mL). The mixture was stirred at roomtemperature for 20 min and transferred to the previously formedpotassium thiolate. The dark solution was then purged with nitrogen andheated to 130° C. for 15 h. After cooling to room temperature, themixture was concentrated under reduced pressure. The crude product wasthen purified by silica gel chromatography with ethyl acetate/petroleumether (1:80˜1:50) to afford 1.8 g (30%) of the title compound as yellowoil. MS (ES, m/z): 414 [M+H]⁺.

Compound 10.6:4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride

Into a 50-mL 3-necked round-bottom flask, was placed a solution of4-(4-(benzylthio)phenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline(250 mg, 0.60 mmol, 1.00 equiv) in acetic acid (8 mL), water (1 mL). Tothe above Cl₂(g) was introduced and the resulting solution was stirredfor 30 min at 25° C. The resulting mixture was concentrated undervacuum. This resulted in 200 mg (85%) of the title compound as a yellowsolid. MS (ES, m/z): 390 [M−HCl+H]⁺.

Compound 10:2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethylphosphonicacid

Following the procedures outlined in Example 1,4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 10.6) was converted to compound 10. Purificationby preparative HPLC gave a TFA salt of the title compound as a whitesolid. ¹H-NMR (CD₃OD, 300 MHz, ppm): 7.93 (d, J=8.4 Hz, 2H), 7.58˜7.51(m, 3H), 6.89 (s, 1H), 4.89˜4.80 (m, 2H), 4.56˜4.51 (m, 1H), 3.95˜3.90(m, 1H), 3.69˜3.65 (m, 1H), 3.21˜3.10 (m 5H), 2.01˜1.89 (m, 2H). MS (ES,min): 479 [M+H]⁺.

Example 11(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)methylphosphonicacid

Compound 11:(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)methylphosphonicacid

Following the procedures outlined in Example 1, compound 11 was madeusing4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 10.6) and diethyl aminomethylphosphonate(intermediate 5.3). Purification by preparative HPLC gave a TFA salt ofthe title compound. ¹H-NMR (300 MHz, DMSO+D₂O, ppm): 7.87 (d, J=8.4 Hz,2H), 7.68 (d, J=1.5 Hz, 1H), 7.48 (d, J=9.4 Hz, 2H), 6.80 (s, 1H),4.74˜4.66 (m, 1H), 4.46˜4.40 (m, 1H), 3.82˜3.77 (m, 1H), 3.69˜3.39 (m,1H), 3.01 (s, 3H), 2.91˜2.74 (m, 2H). MS 465 [M+H]⁺.

Example 123-(4-(6,8-dichloro-2-methyl-1,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)propylphosphonicacid

Compound 12:3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)propylphosphonicacid

Following the procedures outlined in Example 1, compound 12 was madeusing4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 10.6) and 3-diethyl 3-aminopropylphosphonate(intermediate 4.1). Purification by preparative HPLC gave a TFA salt ofthe title compound ¹H-NMR (300 MHz, CD₃OD, ppm): 7.90 (d, J=8.4, 2H),7.55 (s, 1H), 7.46 (d, J=8.1 Hz, 2H), 6.88 (s, 1H), 4.77˜4.82 (m, 1H),4.71 (d, J=16.2 Hz, 1H), 4.47 (d, J=15.9 Hz, 1H), 3.80˜3.86 (m, 1H),3.54˜3.61 (m, 1H), 3.11 (s, 3H), 2.95˜2.99 (m, 2H), 1.53˜1.71 (m, 4H).MS 493 [M+H]⁺.

Example 13(4-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)phenyl)methylphosphonicacid

Compound 13:(4-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)phenyl)methylphosphonicacid

Following the procedures outlined in Example 1, compound 13 was madeusing4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 10.6) and 4-aminobenzylphosphonate (intermediate3.2). Purification by preparative HPLC gave a TFA salt of the titlecompound. ¹H-NMR (300 MHz, DMSO+D₂O, ppm): 7.69 (d, J=8.4 Hz, 2H),7.46˜7.46 (m, 1H), 7.34 (d, J=8.4 Hz, 2H), 7.07 (d, J=7.8 Hz, 2H), 6.94(d, J=8.1 Hz, 2H), 6.71˜6.71 (m, 1H), 4.36˜4.40 (m, 1H), 3.65˜3.80 (m,2H), 2.95˜3.01 (m, 1H), 2.72˜2.79 (m, 3H), 2.41 (s, 3H). MS (ES, m/z):541 [M+H]⁺.

Example 14(4-((4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)methyl)phenyl)methylphosphonic acid

Compound 14:(4-((4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)methyl)phenyl)methylphosphonic acid

Following the procedures outlined in Example 1, compound 14 was madeusing4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 10.6) and 4-(aminomethyl)benzylphosphonate(intermediate 6.1). Purification by preparative HPLC gave a TFA salt ofthe title compound. ¹H-NMR (300 MHz, DMSO+D₂O, ppm): 7.71 (d, J=8.4 Hz,2H), 7.50 (m, 1H), 7.40 (d, J=8.4 Hz, 2H), 7.06˜7.15 (m, 4H), 6.86˜6.87(m, 1H), 4.38˜4.40 (m, 1H), 3.95 (s, 2H), 3.75 (d, J=16.2 Hz, 1H), 3.53(m, 1H), 2.85˜2.92 (m, 3H), 2.69˜2.75 (m, 1H), 2.41 (s, 3H). MS (ES,m/z): 555 [M+H]⁺.

Example 153,3′-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonylazanediyl)dipropanoicacid

Intermediate 15.1:2-((2,4-dichlorobenzyl)(methyl)amino)-1-phenylethanone

Into a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of2-bromo-1-phenylethanone (1 g, 5.05 mmol, 1.00 equiv) in 1,4-dioxane (20mL) and (2,4-dichlorophenyl)-N-methylmethanamine (1.1 g, 5.82 mmol, 1.15equiv). Triethylamine (2 g, 19.80 mmol, 3.92 equiv) was added dropwisewith stirring at 20° C. The resulting solution was stirred for 1 h at20° C. in an oil bath. The solids were filtered out. The resultingmixture was concentrated under vacuum. The residue was applied onto asilica gel column with ethyl acetate/petroleum ether (1:50). Thisresulted in 1.4 g (90%) of the title compound as a yellow oil.

Intermediate 15.2: 2-((2,4-dichlorobenzyl)(methyl)amino)-1-phenylethanol

Into a 250 ml 3-necked roundbottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of2-((2,4-dichlorobenzyl)(methyl)amino)-1-phenylethanone (4.3 g, 14.01mmol, 1.00 equiv) in methanol (50 mL). This was followed by the additionof NaBH₄ (1.5 g, 39.47 mmol, 2.82 equiv) in several batches at 0° C. Theresulting solution was stirred for 30 min at 0° C. in a water/ice bath.The reaction was then quenched by the addition of 20 mL of acetone. Theresulting mixture was concentrated under vacuum. The residue was appliedonto a silica gel column with ethyl acetate/petroleum ether (1:80˜1:20).This resulted in 3.4 g (79%) of the title compound as a white solid.

Intermediate 15.3:6,8-dichloro-2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline

Into a 100-mL 3-necked round-bottom flask, was placed a solution of2-((2,4-dichlorobenzyl)(methyl)amino)-1-phenylethanol (3.4 g, 11.00mmol, 1.00 equiv) in dichloromethane (15 mL). This was followed by theaddition of sulfuric acid (15 mL) dropwise with stirring at 0° C. Theresulting solution was stirred for 2 h at 0° C. in a water/ice bath. ThepH value of the solution was adjusted to 7 with 1M sodium hydroxide. Theresulting solution was extracted with ethyl acetate (3×60 mL) and thecombined organic layers dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was applied onto a silica gelcolumn with petroleum ether:ethyl acetate (80:1). This resulted in 1.6 g(50%) of the title compound as a colorless oil.

Intermediate 15.4:4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Into a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed chlorosulfonic acid (4 mL).This was followed by the dropwise addition of a solution of6,8-dichloro-2-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (1.6 g,5.5 mmol, 1.00 equiv) in dichloromethane (30 mL) at 0° C. The resultingsolution was stirred for 1 h at 0° C. in a water/ice bath and for anadditional 1 h at 25° C. in an oil bath. To this was addedchlorosulfonic acid (16 mL) dropwise at 25° C. The resulting solutionwas stirred for an additional 1 h at 25° C. To the resulting mixture wascooled to 0° C. and aqueous ammonia (120 mL) was added dropwise. Theresulting solution was stirred for an additional 3 h 90° C. in an oilbath. The resulting mixture was concentrated under vacuum. The residuewas dissolved in 20 mL of water. The resulting solution was extractedwith dichloromethane (3×30 mL) and the combined organic layersconcentrated under vacuum. The residue was applied onto a silica gelcolumn with dichloromethane/methanol (100:1). The crude product (0.5 g)was purified by preparative HPLC to give 53 mg (3%) of the titlecompound as a TFA salt. ¹H-NMR (300 MHz, CDCl₃, ppm): 7.89 (1H, d, J=8.4Hz), 7.35 (2H, d, J=8.4 Hz), 7.30 (1H, m), 6.77 (1H, s), 4.87 (1H, s),4.39 (1H, s), 3.69 (2H, m), 2.98 (1H, t), 2.67 (1H, dd), 2.55 (3H, s).MS (ES, m/z): 371 [M+H]⁺.

Intermediate 15.5: dimethyl3,3′-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonylazanediyl)dipropanoate

Into a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 15.4, 100 mg, 0.27 mmol, 1.00 equiv) in acetonitrile (5 mL).Methyl but-3-enoate (40 mg, 0.40 mmol, 1.48 equiv) was added, along with1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 20 mg, 0.13 mmol, 0.49 equiv).The resulting solution was stirred overnight at 25° C. in an oil bath.Removing the solvent under vacuum gave the title compound which was usedwithout further purification.

Compound 15:3,3′-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonylazanediyl)dipropanoicacid

Into a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of Intermediate 15.5(140 mg, 0.26 mmol, 1.0) equiv, theoretical yield) in tetrahydrofuran (5mL) and water (5 mL). LiOH (20 mg, 0.83 mmol, 3.23 equiv) was added andthe resulting solution was stirred for 1 h at room temperature. Theresulting mixture was concentrated under vacuum. The residue was appliedonto a silica gel column with dichloromethane/methanol (100:1˜20:1).This resulted in 0.015 g (11%) of the title compound as a white solid.¹H-NMR (300 MHz, CD₃OD, ppm): 7.84 (d, J=8.1 Hz, 2H), 7.41 (d, J=8.4 Hz,2H), 7.35 (s, 1H), 6.84 (s, 1H), 4.39 (t, 1H), 3.77 (d, 1H), 3.67 (d,1H), 3.45 (m, 1H), 3.33 (m, 4H), 2.69 (d, 1H), 3.0 (m, 1H), 2.47 (m,6H). MS (ES, m/z): 515 [M+H]⁺.

Example 16N,N′,N″-(2,2′,2′-nitrilotris(ethane-2,1-diyl))tris(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 16:N,N′,N″-(2,2′,2″-nitrilotris(ethane-2,1-diyl))tris(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

To a solution of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 1.6) (100 mg, 0.235 mmol) in DMF (1.5 mL) wasadded TEA (94.94 mg, 0.94 mmol) and a solution ofN1,N1-bis(2-aminoethyl)ethane-1,2-diamine (11.45 mg, 0.0783 mmol) in 0.1mL DMF. The reaction was stirred for 40 minutes at which point LCMSindicated no starting material remained. The solvent was removed and theresidue dissolved in 50% acetic acid in water and purified bypreparative HPLC to yield the title compound (25.4 mg) as a TFA salt.¹H-NMR (400 MHz, d₆-DMSO): δ7.77 (s, 1H), 7.75 (s, 1H), 7.64 (s, 1H),7.59 (m, 3H), 6.76 (s, 1H), 4.70 (m, 1H), 4.38 (m, 1H), 3.90 (br m, 8H),3.26 (m, 1H), 3.95 (s, 3H), 2.65 (m, 2H). MS (m/z): 1210.01 (M+H).

Example 17N,N′-(2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 17:N,N′-(2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

To a solution of 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (26.17 mg,0.176 mmol) in chloroform (0.223 mL) at 0° C. was addeddiisopropylethylamine (DIEA, 182 mg, 1.412 mmol) and a solution of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 1.6) (150 mg, 0.353 mmol) in chloroform (0.706mL). The resulting solution was stirred for 10 minutes at which pointthe solvent was removed and the residue taken up in 50%isopropanol/water mixture and purified by preparative HPLC. The titlecompound was obtained (44.5 mg) as a TFA salt. ¹H-NMR (400 MHz, CD₃OD):δ7.87 (d, 1H), 7.78 (d, 1H), 7.64 (t, 1H), 7.55 (d, 1H), 7.51 (d, 1H),6.81 (s, 1H), 4.47 (d, 1H), 3.83 (dd, 1H), 3.59 (t, 1H), 3.43 (m, 2H),3.12 (s, 4H), 3.01 (q, 2H). MS (m/z): 857.17 (M+H).

Example 18N,N′-(1,4-phenylenebis(methylene))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 18:N,N′-(1,4-phenylenebis(methylene))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Following the procedures outlined in Example 17, compound 18 was madeusing 1,4-phenylenedimethanamine as the amine. Purification bypreparative HPLC gave the title compound as a TFA salt. ¹H-NMR (400 MHz,CD₃OD): δ 7.87 (d, 2H), 7.67 (s, 2H), 7.52 (m, 4H), 7.49 (d, 2H), 7.09(s, 4H), 6.82 (s, 2H), 4.78 (m, 7H), 4.43 (d, 2H), 4.00 (s, 4H), 3.82(dd, 2H), 3.51 (t, 2H), 3.11 (s, 6H). MS (m/z): 845.03 (M+H).

Example 19N,N′-(butane-1,4-diyl)bis(3-(6,8-dichlor-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 19:N,N′-(butane-1,4-diyl)bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Following the procedures outlined in Example 17, compound 19 was madeusing butane-1,4-diamine as the amine. Purification by preparative HPLCgave the title compound as a TFA salt. ¹H-NMR (400 MHz, CD₃OD): δ 7.85(d, 2H), 7.80 (s, 2H), 7.63 (t, 2H), 7.54 (t, 4H), 6.82 (s, 2H), 4.49(d, 1H), 3.88 (dd, 2H), 3.58 (t, 2H), 3.14 (s, 6H), 2.81 (m, 4H), 1.42(m, 4H). MS (m/z): 797, 19 (M+H).

Example 20N,N′-(dodecane-1,12-diyl)bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 20:N,N′-(dodecane-1,12-diyl)bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Following the procedures outlined in Example 17, compound 20 was madeusing dodecane-1,12-diamine as the amine. Purification by preparativeHPLC gave the title compound as a TFA salt. ¹H-NMR (400 MHz, CD₃OD):δ7.85 (d, 2H), 7.71 (s, 2H), 7.63 (t, 2H), 7.54 (m, 4H), 6.81 (s, 2H),4.74 (m, 2H), 4.51 (d, 2H), 3.86 (dd, 2H), 3.29 (t, 2H), 3.13 (s, 7H),2.79 (t, 4H), 1.39 (m, 4H), 1.22 (m, 20H). MS (m/z): 909.28 (M+H).

Example 21N,N′,N″,N″′-(3,3′,3″,3″′-(butane-1,4-diylbis(azanetriyl))tetrakis(propane-3,1-diyl))tetrakis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 21:N,N′,N″,N″′-(3,3′,3″,3′″-(butane-1,4-diylbis(azanetriyl))tetrakis(propane-3,1-diyl))tetrakis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

To a solution of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 1.6) (150 mg, 0.352 mmol) in THF/H₂O (0.704 mL,50% v/v) was added DIEA (181.6 mg, 1.41 mmol) and finallyN1,N1′-(butane-1,4-diyl)bis(N1-(3-aminopropyl)propane-1,3-diamine)(27.94 mg, 0.08825 mmol). The reaction mixture was stirred vigorouslyfor 1 hour at which point the solvent was removed. The resulting residuewas brought up in 50% acetonitrile/water and purified by preparativeHPLC to give the title compound (117 mg) as a TFA salt. ¹H-NMR (400 MHz,CD₃OD): δ7.85 (d, 2H), 7.78 (s, 2H), 7.62 (t, 2H), 7.36 (m, 4H), 6.79(s, 2H), 4.78 (m, 4H), 4.47 (d, 2H), 3.86 (dd, 2H), 3.55 (t, 2H), 3.12(s, 6H), 2.94 (m, 4H), 1.90 (m, 4H), 1.85 (m, 2H). MS (m/z): 1732.90(M+H).

Example 22N,N′-(butane-1,4-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 22:N,N′-(butane-1,4-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

To a solution of4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 10.6) (150 mg, 0.353 mmol) in chloroform (0.706mL) was added DIEA (182 mg, 1.412 mmol) and a solution ofbutane-1,4-diamine (15.5 mg, 0.176 mmol) in chloroform (0.176 mL). Thereaction was stirred overnight at which point the solvent was removedand the resulting residue brought up in 50% IPA/H₂O. Purification bypreparative HPLC gave the title compound (18.4 mg) as a TFA salt. ¹H-NMR(400 MHz, CD₃OD): δ7.86 (d, 4H), 7.53 (s, 2H), 7.45 (d, 4H), 6.84 (s,2H), 4.73 (m, 3H), 4.46 (d, 2H), 3.86 (dd, 2H), 3.57 (t, 2H), 3.12 (s,6H), 2.84 (m, 4H), 1.41 (m, 4H). MS (m/z): 797.15 (M+H).

Example 23N,N′-(dodecane-1,12-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 23:N,N′-(dodecane-1,12-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Following the procedures outlined in Example 22, compound 23 was madeusing dodecane-1,12-diamine as the amine. Purification by preparativeHPLC gave the title compound as a TFA salt. ¹H-NMR (400 MHz, CD₃OD):7.89 (d, 4H), 7.54 (m, 2H), 7.42 (m, 4H), 6.82 (s, 2H), 4.85 (m, 3H),4.72 (d, 2H), 3.85 (dd, 2H), 3.59 (t, 2H), 3.13 (m, 8H), 2.85 (m, 4H),1.89 (m, 5H), 1.33 (m, 23H). MS (m/z): 909.21 (M+H).

Example 24N,N′,N″-(2,2′,2″-nitrilotris(ethane-2,1-diyl))tris(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 24:N,N′,N″-(2,2′,2″-nitrilotris(ethane-2,1-diyl))tris(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

To a solution of4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 10.6) (150 mg, 0.353 mmol) in THF/H₂O solution(50% v/v, 0.704 mL) was added DIEA (182.2 mg, 1.412 mmol) andN1,N1-bis(2-aminoethyl)ethane-1,2-diamine (17.0 mg, 0.116 mmol). Thereaction was stirred vigorously at room temperature for 40 minutes atwhich point the solvent was removed. The resulting residue was dissolvedin acetonitrile/water (50% v/v) and purified by preparative HPLC to givethe title compound (57.6 mg) as a TFA salt. ¹H-NMR (400 MHz, CD₃OD):7.94 (d, 6H), 7.51 (t, 9H), 6.83 (s, 3H), 4.78 (m, 6H), 4.45 (d, 3H),3.83 (dd, 3H), 3.49 (t, 3H), 3.30 (m, 6H), 3.29 (m, 21H), 3.12 (s, 9H).MS (m/z): 1208.09 (M+H).

Example 25N,N′,N″,N″′-(3,3′,3″,3″-(butane-1,4-diylbis(azanetriyl))tetrakis(propane-3,1-diyl))tetrakis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 25:N,N′,N″,N″′-(3,3′,3″,3″′-(butane-1,4-diylbis(azanetriyl))tetrakis(propane-3,1-diyl))tetrakis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Following the procedure outlined in Example 24, Compound 25 was madeusing N1,N1′-(butane-1,4-diyl)bis(N1-(3-aminopropyl)propane-1,3-diamine)as the amine. Purification by preparative HPLC gave the title compoundas a TFA salt. ¹H-NMR (400 MHz, CD₃OD): 7.88 (d, 8H), 7.51 (s, 4H), 7.48(d, 8H), 6.81 (s, 4H), 4.75 (m, 8H), 4.47 (d, 4H), 3.85 (dd, 4H), 3.58(t, 4H), 3.13 (s, 12H), 2.98 (t, 8H), 1.97 (m, 8H), 1.88 (m, 4H). MS(m/z): 1733.02 (M+H).

Example 26N,N′-(1,4-phenylenebis(methylene))bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 26:N,N′-(1,4-phenylenebis(methylene))bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Following the procedure outlined in Example 24, compound 26 was madeusing 1,4-phenylenedimethanamine as the amine. Purification bypreparative HPLC gave the title compound as a TFA salt. ¹H-NMR (400 MHz,CD₃OD): 7.76 (d, 4H), 7.54 (s, 2H), 7.39 (d, 4H), 7.08 (s, 4H), 6.82 (s,2H), 4.72 (m, 3H), 4.47 (d, 2H), 4.07 (s, 4H), 3.88 (dd, 2H), 3.61 (t,2H), 3.16 (s, 6H). MS (m/z): 845.07 (M+H).

Example 27N,N′-(2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 27:N,N′-(2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Following the procedure outlined in Example 24, compound 27 was madeusing 2,2′-(ethane-1,2-diylbis(oxy))diethanamine as the amine.Purification by preparative HPLC gave the title compound as a TFA salt.¹H-NMR (400 MHz, CD₃OD): 7.89 (d, 4H), 7.52 (s, 2H), 7.47 (d, 4H), 6.82(s, 2H), 4.77 (m, 4H), 4.47 (d, 2H), 3.86 (dd, 2H), 3.59 (t, 2H), 3.43(t, 8H), 3.13 (s, 6H), 3.06 (t, 4H). MS (m/z): 857.15 (M+H).

Example 28N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Intermediate 28.1 N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

To a solution of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 1.6) (600 mg, 1.41 mmol) in chloroform (2.82 mL)was added DIEA (545.7 mg, 4.24 mmol) and2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (616.3 mg, 2.82 mmol).The reaction was stirred overnight at which point the mixture wasdiluted with 50 mL DCM and washed with NaHCO₃(50 mL). The aqueous layerwas extracted with DCM (2×50 mL) and the combined organic fractionswashed with water (200 mL), brine (200 mL), and dried over Na₂SO₄.Removing the solvent gave the title compound as an oil which was usedwithout further purification.

Compound 28:N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 28.1) (1.035 g, assume 1.41 mmol) was dissolved in a 10:1THF:water solution (26.5 mL) and placed under N₂. PMe₃ (165 mg, 2.18mmol) was added and the reaction stirred overnight. The solvent wasremoved and the resulting residue brought up in EtOAc (100 mL) andwashed with NaHCO₃(100 mL) and brine (100 mL). After drying the organiclayer over Na₂SO₄, the solvent was removed to give 446 mg of the titlecompound (58% over two steps) as an oil. A portion of the crude productwas purified by preparative HPLC to give the title compound as a TFAsalt. ¹H-NMR (400 mHz, CD₃OD) δ 7.87 (m, 1H), 7.73 (m, 1H), 7.67 (t,j=7.7 Hz, 1H), 7.54 (m, 2H), 6.82 (s, 1H), 4.8-4.6 (m, 4H), 4.46 (m,1H), 3.86 (m, 1H), 3.69 (m, 2H), 3.66 (s, 3H), 3.61 (m, 2H), 3.55 (m,2H), 3.12 (m, 4H), 3.03 (t, j=5.4 Hz, 1H). MS (m/z): 546.18 (M+H).

Example 29N1,N8-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)octanediamide

Compound 29:N1,N8-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)octanediamide

To a solution ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(compound 28) (54.5 mg, 0, mmol) in DMF (0.20 mL) was added DIEA (15.5mg, 0.12 mmol) and bis(2,5-dioxopyrrolidin-1-yl) octanedioate (18.4 mg,0.05 mmol). The reaction was stirred at room temperature for 3 hours atwhich point an additional 0.03 mmol of compound 28 was added. After afurther hour the solvent was removed and the resulting residue dissolvedin acetonitrile/water (1:1) and purified by preparative HPLC to give thetitle compound (17.4 mg) as a TFA salt. ¹H-NMR (400 MHz, CD₃OD): 7.89(d, 2H), 7.78 (s, 2H), 7.64 (t, 2H), 7.52 (m, 4H), 6.83 (s, 2H), 4.81(m, 4H), 4.45 (d, 2H), 3.89 (dd, 2H), 3.61 (m, 18H), 3.55 (m, 10H), 3.47(m, 5H), 3.33 (m, 5H), 3.14 (s, 7H), 3.04 (t, 4H), 2.16 (t, 4H), 1.55(m, 4H), 1.29 (m, 4H). MS (m/z): 1231.87 (M+H).

Example 302-(N-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)ethylphosphonicacid

Intermediate 30.1: 1-(4-aminophenyl)ethanone

Into a 100-mL 3-necked round-bottom flask, was placed a solution of1-(4-nitrophenyl)ethanone (6 g, 36.36 mmol, 1.00 equiv) in ethanol (100mL), water (15 mL). This was followed by the addition of NH₄Cl (3.85 g,72.64 mmol, 2.00 equiv) in several batches. To this was added Fe (10.18g, 181.79 mmol, 5.00 equiv) in several batches, while the temperaturewas maintained at reflux. The resulting mixture was heated to reflux for2 h. The solids were filtered out and the resulting filtrate wasconcentrated under vacuum. The residue was diluted with 50 mL of water.The resulting solution was extracted with 3×50 mL of ethyl acetate andthe organic layers combined and dried over anhydrous sodium sulfate andconcentrated under vacuum to give 3.1 g (60%) of1-(4-aminophenyl)ethanone as a yellow solid.

Intermediate 30.2: N-(4-acetylphenyl)acetamide

Into a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of1-(4-aminophenyl)ethanone (3.1 g, 22.96 mmol, 1.00 equiv) indichloromethane (30 mL), triethylamine (4.64 g, 45.94 mmol, 2.00 equiv).This was followed by the addition of acetyl chloride (1.79 g, 22.95mmol, 1.00 equiv) dropwise with stirring at 0° C. The resulting solutionwas stirred for 30 min at 0° C. The reaction was then quenched by theaddition of 2 mL of water. The resulting mixture was washed with 3×50 mLof saturated aqueous sodium chloride. The mixture was dried overanhydrous sodium sulfate and concentrated under vacuum to give 3.0 g(74%) of N-(4-acetylphenyl)acetamide as a white solid.

Intermediate 30.3: N-(4-(2-bromoacetyl)phenyl)acetamide

Into a 100-mL 3-necked round-bottom flask, was placed a solution ofN-(4-acetylphenyl)acetamide (1 g, 5.65 mmol, 1.00 equiv) in acetic acid(10 mL). This was followed by the addition of a solution of bromine (910mg, 5.69 mmol, 1.01 equiv) in acetic acid (2 mL) dropwise with stirringat 50° C. The resulting solution was stirred for 1.5 h at 50° C. Thereaction was then quenched by the addition of 100 mL of water/ice. Thesolids were collected by filtration and dried under vacuum. Thisresulted in 0.5 g (33%) of N-(4-(2-bromoacetyl)phenyl)acetamide as awhite solid.

Intermediate 30.4:N-(4-(2-((2,4-dichlorobenzyl)(methyl)amino)acetyl)phenyl)acetamide

Into a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution ofN-(4-(2-bromoacetyl)phenyl)acetamide (1 g, 3.91 mmol, 1.00 equiv) in1,4-dioxane (40 mL). This was followed by the addition of triethylamine(1.58 g, 15.64 mmol, 4.00 equiv) dropwise with stirring at 20° C. Tothis was added (2,4-dichlorophenyl)-N-methylmethanamine (880 mg, 4.63mmol, 1.19 equiv) dropwise with stirring at 20° C. The resultingsolution was stirred for 4 h at 20° C. The solids were filtered out. Theresulting mixture was concentrated under vacuum to give 1.5 g (84%) ofN-(4-(2-((2,4-dichlorobenzyl)(methyl)amino)acetyl)phenyl)acetamide as awhite solid.

Intermediate 30.5:N-(4-(2-((2,4-dichlorobenzyl)(methyl)amino)-1-hydroxyethyl)phenyl)acetamide

Into a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution ofN-(4-(2-((2,4-dichlorobenzyl)(methyl)amino)acetyl)phenyl)acetamide (1.5g, 4.11 mmol, 1.00 equiv) in methanol (20 mL). This was followed by theaddition of NaBH₄ (300 mg, 7.89 mmol, 2.06 equiv) in several batches at0-5° C. The resulting solution was stirred for 2 h at 0-5° C. Thereaction was then quenched by the addition of 5 mL of acetone. Theresulting mixture was concentrated under vacuum. The residue was appliedonto a silica gel column with ethyl acetate/petroleum ether (1:10-1:5).This resulted in 1.2 g (76%) ofN-(4-(2-((2,4-dichlorobenzyl)methyl)amino)-1-hydroxyethyl)phenyl)acetamideas yellow oil.

Intermediate 30.6:N-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)acetamide

Into a 100-mL 3-necked round-bottom flask, was placed a solution ofN-(4-(2-((2,4-dichlorobenzyl)(methyl)amino)-1-hydroxyethyl)phenyl)acetamide(500 mg, 1.36 mmol, 1.00 equiv) in dichloromethane (3 mL). This wasfollowed by the addition of sulfuric acid (3 mL) dropwise with stirringat 0° C. The resulting solution was stirred for 5 h at 0-5° C. Thereaction was then quenched by the addition of 20 mL of water/ice. The pHvalue of the solution was adjusted to 7-8 with sodium hydroxide. Theresulting solution was extracted with 3×20 mL of ethyl acetate and theorganic layers combined and concentrated under vacuum. The residue wasapplied onto a silica gel column with ethyl acetate/petroleum ether(1:10-1:5). This resulted in 25 mg (5%) ofN-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)acetamideas a white solid. ¹H-NMR (300 HMz, CDCl₃, ppm): δ 7.46-7.49 (2H, d,J=8.4 Hz), 7.23-7.29 (1H, m), 7.12-7.15 (2H, d, J=8.4 Hz), 6.80 (1H, s),4.314 (1H, s), 3.92 (1H, d), 3.58-3.63 (1H, d), 3.06 (1H, s), 2.61-2.68(1H, m), 2.57 (3H, s), 2.20 (3H, s). MS (ES, m/z): 349 [M+H]⁺.

Intermediate 30.7:4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenamine

Into a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution ofN-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)acetamide(2 g, 5.73 mmol, 1.00 equiv) in ethanol (20 mL). This was followed bythe addition of sodium methanolate (5 g, 92.59 mmol, 16.16 equiv) inseveral batches, while the temperature was maintained at reflux. Theresulting solution was heated to reflux overnight. The reaction was thenquenched by the addition of 50 mL of water/ice. The resulting solutionwas extracted with 3×50 mL of ethyl acetate and the organic layerscombined and concentrated under vacuum. This resulted in 1.5 g (85%) of4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenamineas yellow oil. ¹H-NMR (300 MHz, DMSO, ppm): δ 7.42-7.42 (1H, d, J=1.5Hz), 6.83-6.86 (2H, d, J=8.1 Hz), 6.78-6.78 (1H, d, J=1.2 Hz), 6.48-6.51(2H, d, J=8.4 Hz), 4.98 (2H, s), 4.02-4.06 (1H, m), 3.62-3.67 (1H, d,J=16.2 Hz), 3.43-3.48 (1H, d, J=15.9 Hz), 2.80-2.86 (1H, m), 2.37 (3H,s). MS (ES, m/z): 307 [M+H]1.

Intermediate 30.8: diethyl 2-(chlorosulfonylamino)ethylphosphonate

Into a 100-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of sulfuryl dichloride(1.1 g, 8.15 mmol, 1.47 equiv) in dichloromethane (10 mL). This wasfollowed by the addition of a solution of diethyl2-aminoethylphosphonate (intermediate 1.9) (1.0 g, 5.52 mmol, 1.00equiv) and triethylamine (800 mg, 7.92 mmol, 1.43 equiv) indichloromethane (20 mL) dropwise with stirring at 0° C. The resultingsolution was stirred for 2 h at 0° C. The reaction was then quenched bythe addition of ice water. The organic layer was washed with saturatedsodium chloride (20 mL), dried over anhydrous sodium sulfate andconcentrated under vacuum. This resulted in 0.5 g (crude) of the titlecompound as a colorless oil.

Intermediate 30.9: diethyl2-(N-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)ethylphosphonate

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed diethyl2-(chlorosulfonylamino)ethylphosphonate (intermediate 30.8) (670 mg,2.40 mmol, 1.47 equiv),4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenamine(intermediate 30.7) (500 mg, 1.63 mmol, 1.00 equiv),N-ethyl-N-isopropylpropan-2-amine (400 mg, 3.10 mmol, 1.91 equiv) inacetonitrile (20 mL). The resulting solution was stirred for 3 h at 60°C. The resulting mixture was concentrated under vacuum and the residuewas applied to a silica gel column and eluted withdichloromethane/methanol (20:1). This resulted in 150 mg (16%) of thetitle compound as a light yellow solid.

Compound 30:2-(N-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)ethylphosphonicacid

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of diethyl2-(N-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)ethylphosphonate(100 mg, 0.18 mmol, 1.00 equiv) in dichloromethane (5 mL) andbromotrimethylsilane (275 mg, 1.80 mmol, 9.89 equiv). The resultingsolution was stirred overnight at 39° C. The resulting mixture wasconcentrated under vacuum and the residue was dissolved indichloromethane (5 mL). This was followed by the addition of a solutionof sodium hydroxide (14.5 mg, 0.36 mmol, 2.00 equiv) in methanol (0.2mL) dropwise with stirring. The solids were collected by filtration anddried under reduced pressure. This gave 40 mg (40%) of a sodium salt ofthe title compound as a white solid. ¹H-NMR (300 MHz, d₆-DMSO, ppm): δ9.78 (1H, brs), 7.54 (1H, s), 7.47 (1H, brs), 7.09-7.17 (4H, m), 6.82(1H, s), 4.31 (1H, brs), 3.88 (2H, brs), 3.13 (1H, brs), 3.04 (2H, brs),2.90 (1H, brs), 2.58 (3H, s), 1.65-1.77 (2H, m). MS (m/z): 494 [M+H]⁺.

Example 312-(N-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)ethylphosphonicacid

Intermediate 31.1: 2-bromo-1-(3-nitrophenyl)ethanone

Into a 500-mL 3-necked round-bottom flask, was placed a solution of1-(3-nitrophenyl)ethanone (50 g, 303.03 mmol, 1.00 equiv) in acetic acid(300 mL), Br₂ (53.5 g, 331.6 mmol, 1.00 equiv). The resulting solutionwas stirred for 2 h at 60° C. in an oil bath. The reaction was thenquenched by the addition of ice and the solids were collected byfiltration. The crude product was re-crystallized from ethylacetate/petroleum ether in the ratio of 1:10. This resulted in 25 g(34%) of 2-bromo-1-(3-nitrophenyl)ethanone as a white solid.

Intermediate 31.2:2-((2,4-dichlorobenzyl)methyl)amino)-1-(3-nitrophenyl)ethanone

Into a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of2-bromo-1-(3-nitrophenyl)ethanone (2 g, 8.23 mmol, 1.00 equiv),triethylamine (3.4 g, 4.00 equiv),(2,4-dichlorophenyl)-N-methylmethanamine (1.9 g, 10.05 mmol, 1.20equiv), 1,4-dioxane (50 mL). The resulting solution was stirred for 2 hat room temperature at which time it was judged to be complete by LCMS.The mixture was concentrated under vacuum and the residue was appliedonto a silica gel column with ethyl acetate/petroleum ether(1:100˜1:50). This resulted in 1.5 g (50%) of2-((2,4-dichlorobenzyl)methyl)amino)-1-(3-nitrophenyl)ethanone as ayellow solid.

Intermediate 31.3:2-((2,4-dichlorobenzyl)(methyl)amino)-1-(3-nitrophenyl)ethanol

Into a 500-mL 3-necked round-bottom flask, was placed a solution of2-((2,4-dichlorobenzyl)(methyl)amino)-1-(3-nitrophenyl)ethanone (28 g,1.00 equiv, Crude) in methanol (280 mL), NaBH₄ (6.38 mg, 0.17 mmol, 2.00equiv). The resulting solution was stirred for 0.5 h at 0° C. Thereaction progress was monitored by LCMS. The reaction was then quenchedby the addition of 10 mL of acetone. The resulting mixture wasconcentrated under vacuum. The residue was applied onto a silica gelcolumn with ethyl acetate/petroleum ether (1:10-1:5). This resulted in14 g of 2-((2,4-dichlorobenzyl)(methyl)amino)-1-(3-nitrophenyl)ethanolas a yellow solid.

Intermediate 31.4:6,8-dichloro-2-methyl-4-(3-nitrophenyl)-1,2,3,4-tetrahydroisoquinoline

Into a 500-mL 3-necked round-bottom flask, was placed a solution of2-((2,4-dichlorobenzyl)(methyl)amino)-1-(3-nitrophenyl)ethanol (14 g,39.55 mmol, 1.00 equiv) in dichloromethane (140 mL), sulfuric acid (140mL). The resulting solution was stirred overnight at room temperature.The reaction progress was monitored by LCMS. The resulting solution wasdiluted with 100 mL of ice. The pH value of the solution was adjusted to8-9 with sat. sodium hydroxide (100 mL). The resulting solution wasextracted with 2×500 mL of ethyl acetate and the organic layers combinedand dried over sodium sulfate. The residue was applied onto a silica gelcolumn with ethyl acetate/petroleum ether (1:10-1:5). This resulted in 7g (51%) of6,8-dichloro-2-methyl-4-(3-nitrophenyl)-1,2,3,4-tetrahydroisoquinolineas a yellow solid.

Intermediate 31.5:3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenamine

Into a 100-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed6,8-dichloro-2-methyl-4-(3-nitrophenyl)-1,2,3,4-tetrahydroisoquinoline(200 mg, 0.59 mmol, 1.00 equiv), Fe (360 mg, 6.43 mmol, 8.60 equiv),hydrogen chloride (0.02 mL), ethanol (0.6 mL), water (0.2 mL). Theresulting solution was stirred for 0.5 h at 80° C. in an oil bath. Thesolids were filtered out. The resulting mixture was concentrated undervacuum. This resulted in 0.2 g (crude) of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenamineas yellow oil.

Compound 31:2-(N-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)ethylphosphonicacid

Following the procedures outlined in Example 30, substituting3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline(intermediate 31.5) for4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline gavethe title compound as a sodium salt. ¹H-NMR (300 MHz. D₂O+DMSO-d₆, ppm):δ 7.67 (s, 1H), 7.33 (t, J=8.1 Hz, 1H), 7.07-7.15 (m, 2H), 6.81-6.86 (m,2H), 4.39-4.66 (m, 3H), 3.75-3.81 (m, 1H), 3.45-3.50 (m, 1H), 3.02-3.08(m, 5H), 1.67-1.78 (m, 2H). MS (ES, m/z): 494.0 [M+H]⁺.

Example 323-(N-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)propylphosphonicacid

Compound 32:3-(N-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)propylphosphonicacid

Following the procedures outlined in Example 30, substituting 3-diethyl3-aminopropylphosphonate (intermediate 4.1) for diethyl2-aminoethylphosphonate gave the title compound as a sodium salt. ¹H-NMR(300 MHz, CD₃OD, ppm): δ 7.47 (s, 1H), 7.28 (s, 4H), 6.81 (s, 1H),4.73-4.77 (m, 2H), 4.57 (m, 1H), 3.81 (s, 1H), 3.66 (s, 1H), 3.18 (s,3H), 3.06 (s, 2H), 1.74 (m, 4H), 1.20-1.35 (m, 1H). MS (ES, m/z): 508[M+H]⁺

Example 333-(N-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)propylphosphonicacid

Compound 33:3-(N-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)sulfamoylamino)propylphosphonicacid

Following the procedures outlined in Example 30, substituting 3-diethyl3-aminopropylphosphonate (intermediate 4.1) for diethyl2-aminoethylphosphonate and3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline(intermediate 31.5) for4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline gavethe title compound as a sodium salt. ¹H-NMR (300 MHz, CD₃OD, ppm): δ7.54 (s, 1H), 7.38 (s, 1H), 7.25 (s, 1H), 7.11 (s, 1H), 6.94 (m, 2H),4.66 (s, 1H), 4.55-4.51 (m, 1H), 3.89 (s, 1H), 3.65 (m, 2H), 3.18 (s,3H), 3.05 (s, 2H), 1.71 (m, 4H). MS (ES, m/z): 508 [M+H]⁺.

Example 34(2S)-2-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)succinicacid

Intermediate 34.1: (2S)-dimethyl2-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)succinate

Into a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenamine(intermediate 30.7) (200 mg, 0.65 mmol, 1.00 equiv) in dichloromethane(10 mL), triethylamine (1.2 mL). This was followed by the addition ofbis(trichloromethyl) carbonate (200 mg, 0.67 mmol, 1.03 equiv) slowlywith stirring at 0-5° C. The resulting solution was stirred for 1 h atroom temperature. To this was added triethylamine (1 mL) followed by(S)-dimethyl 2-aminosuccinate (200 mg, 1.24 mmol, 1.91 equiv) in severalbatches. The resulting solution was stirred for 2 h at room temperature.The resulting mixture was concentrated under vacuum and the residue wasapplied onto a silica gel column and eluted with ethyl acetate/petroleumether (1:10-1:5). This resulted in 50 mg (15%) of (2S)-dimethyl2-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)succinateas yellow oil.

Compound 34:(2S)-2-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)succinicacid

Into a 50-mL round-bottom flask, was placed a solution of (2S)-dimethyl2-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)succinate(100 mg, 0.20 mmol, 1.00 equiv) in methanol (5 mL), water (1 mL), sodiumhydroxide (30 mg, 0.75 mmol, 3.71 equiv). The resulting solution wasstirred for 3 h at room temperature and then concentrated under vacuum.The pH of the solution was adjusted to 3-4 with 1N hydrochloric acid.The solids were collected by filtration and the residue was lyophilized.This resulted in 16 mg (16%) of(2S)-2-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)succinicacid as a white solid. ¹H-NMR (300 MHz, DMSO, ppm): δ 8.98 (s, 1H), 7.66(s, 1H), 7.38-7.44 (d, J=17.1 Hz, 2H), 7.12-7.15 (d, J=−8.4 Hz, 2H),6.78 (s, 1H), 6.60-6.63 (s, 1H), 4.48-4.54 (m, 4H), 3.63-3.66 (s, 2H),3.01 (s, 1H), 2.51-2.84 (m, 2H). MS (ES, m/z): 466 [M+H]⁺.

Example 35(2S)-2-(3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)succinicacid

Compound 35:(2S)-2-(3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)succinicacid

Following the procedures outlined in Example 34, substituting3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline(intermediate 31.5) for4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)anilinegave, after purification by preparative HPLC, the title compound as aTFA salt. ¹H-NMR (300 MHz, DMSO, ppm): δ 8.88 (s, 1H), 7.54 (s, 1H),7.31-7.18 (m, 3H), 6.83-6.78 (m, 2H), 6.53-6.51 (m, 1H), 4.49-4.47 (m,1H), 4.29 (m, 1H), 3.87 (m, 2H), 3.32 (m, 2H), 2.76-2.59 (m, 2H), 2.50(s, 3H). MS 466 [M+H]⁺.

Example 36(2S)-2-(34(6,8-dichloro-2-methyl-1,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)pentanedioicacid

Compound 36:(2S)-2-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)ureido)pentanedioicacid

Following the procedures outlined in Example 34, substituting(S)-diethyl 2-aminopentanedioate for (S)-dimethyl 2-aminosuccinate gavethe title compound. ¹H-NMR (300 MHz, DMSO, ppm) δ 12.32 (s, 2H), 8.63(s, 1H), 7.47 (s, 1H), 7.30-7.33 (d, J=8.1 Hz, 2H), 7.06-7.09 (d, J=5.4Hz, 2H), 6.79 (s, 1H), 6.45-6.48 (d, J=8.1 Hz, 1H), 4.19-4.20 (s, 2H),3.68 (s, 2H), 2.95 (s, 1H), 2.68 (s, 1H), 2.45 (s, 3H), 2.27-2.30 (s,2H), 1.99-2.02 (s, 1H), 1.76-7.78 (s, 1H). MS (ES, m/z): 480 [M+H]⁺.

Example 37(2S)-2-(3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)pentanedioicacid

Compound 37:(2S)-2-(3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)phenyl)ureido)pentanedioicacid

Following the procedures outlined in Example 34, substituting3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline(intermediate 31.5) for4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline and(S)-diethyl 2-aminopentanedioate for (S)-dimethyl 2-aminosuccinate gave,after purification by preparative HPLC, the title compound as a TFAsalt. ¹H-NMR (300 MHz, DMSO-d₆, ppm): δ 8.74 (s, 1H), 7.67 (s, 1H), 7.42(m, 1H), 7.27-7.25 (m, 2H), 6.79 (m, 2H), 6.52-6.49 (m, 1H), 4.63-4.58(m, 1H), 4.44 (m, 2H), 4.20-4.16 (m, 1H), 3.72-3.64 (m, 2H), 2.99 (s,3H), 2.34-2.27 (m, 2H), 2.01-1.97 (m, 2H), 1.82-1.77 (m, 2H). MS 480[M+H]⁺.

Example 38(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)methylphosphonicacid

Intermediate 38.1: 4-nitrophenyl4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylcarbamate

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenamine(intermediate 30.7) (300 mg, 0.98 mmol, 1.00 equiv) in dichloromethane(10 mL). This was followed by the addition of 4-nitrophenylchloroformate (230 mg, 1.14 mmol, 1.20 equiv) in several batches at roomtemperature. The resulting solution was stirred for 3 h at roomtemperature. The solids were collected by filtration. This resulted in0.3 g (65%) of 4-nitrophenyl4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylcarbamateas a yellow solid.

Intermediate 38.2: diethyl(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)methylphosphonate

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of 4-nitrophenyl4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylcarbamate(200 mg, 0.42 mmol, 1.00 equiv) in N,N-dimethylformamide (6 mL), asolution of diethyl aminomethylphosphonate (144 mg, 0.63 mmol, 1.50equiv) in N,N-dimethylformamide (1 mL) and triethylamine (64 mg). Theresulting solution was stirred overnight at room temperature. Thereaction was then quenched by the addition of 10 mL of water. Theresulting solution was extracted with 3×10 mL of ethyl acetate and theorganic layers combined and dried over anhydrous sodium sulfate andconcentrated under vacuum. This resulted in 40 mg (17%) of diethyl(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)methylphosphonateas a solid.

Compound 38:(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)methylphosphonicacid

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of diethyl(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)methylphosphonate(40 mg, 0.08 mmol, 1.00 equiv) in dichloromethane (5 mL) andbromotrimethylsilane (0.15 mL). The resulting solution was stirredovernight at room temperature. The resulting mixture was concentratedunder vacuum. To the above was added methanol (5 mL) and sodiumhydroxide (5 mg). The resulting mixture was stirred 0.5 h at roomtemperature. The solids were collected by filtration and the residue waslyophilized. This resulted in 17.4 mg (42%) a sodium salt of the titlecompound as a yellow solid. ¹H-NMR (300 MHz, CD₃OD+DCl, ppm): δ7.46-7.49 (m, 3H), 7.20-7.23 (d, J=8.7 Hz, 2H), 6.80 (s, 1H), 4.77-4.83(d, J=15.9 Hz, 1H), 4.65-4.71 (m, 1H), 4.50-4.55 (d, J=16.2 Hz, 1H),3.79-3.85 (m, 1H), 3.56-3.69 (m, 3H), 3.32 (s, 3H). MS (ES, m/z): 444[M+H]⁺.

Example 39(3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)methylphosphonicacid

Compound 39:(3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)methylphosphonicacid

Following the procedures outlined in Example 38, substituting3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline(intermediate 31.5) for4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline gavethe title compound as a sodium salt. ¹H-NMR (300 MHz, CD₃OD, ppm): δ7.47 (s, 1H), 7.37 (m, 3H), 6.96 (m, 1H), 6.82 (s, 1H), 4.81 (m, 1H),4.70 (m, 1H), 4.54 (m, 1H), 3.83 (m, 1H), 3.65 (m, 3H), 3.19 (s, 3H).

Example 402-(3-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)propyl)malonicacid

Intermediate 40.1: ethyl3-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)propanoate

Following the procedures outlined in Example 34, substituting ethyl3-aminopropanoate for (S)-dimethyl 2-aminosuccinate gave the titlecompound as a yellow oil.

Intermediate 40.2:3-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)propanoicacid

Into a 50-mL round-bottom flask, was placed a solution of ethyl3-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)propanoate(150 mg, 0.33 mmol, 1.00 equiv) in methanol (10 mL), water (2 mL) andsodium hydroxide (80 mg, 2.00 mmol). The resulting solution was stirredfor 2 h at 25° (and the resulting mixture was concentrated under vacuum.The pH value of the solution was adjusted to 7-8 with hydrogen chloride.The resulting solution was extracted with chloroform (3×10 ml) and theorganic layers combined and dried over sodium sulfate. This resulted in31.5 mg (22%) of3-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)propanoicacid as a white solid. ¹H-NMR (300 MHz. DMSO, ppm): δ 8.56 (1H, s), 7.45(1H, s), 7.29-7.32 (2H, d, J=8.1 Hz), 7.04-7.07 (2H, d, J=8.4 Hz), 6.79(1H, s), 6.21 (1H, s), 4.16 (1H, m), 3.56-3.58 (2H, d, J=5.4 Hz),3.27-3.29 (2H, d, J=6 Hz), 2.82-2.87 (1H, m), 2.59 (2H, s), 2.38-2.40(4H, m). MS (ES, m/z): 422 [M+H]⁺.

Intermediate 40.3:1-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)-3-(3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-3-oxopropyl)urea

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of3-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)propanoicacid (200 mg, 0.47 mmol, 1.00 equiv) in dichloromethane (20 mL),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (136 mg,0.71 mmol, 1.50 equiv) and 4-dimethylaminopyridine (115 mg, 0.94 mmol,1.99 equiv). This was followed by the addition of a solution of2,2-dimethyl-1,3-dioxane-4,6-dione (102 mg, 0.71 mmol, 1.49 equiv) indichloromethane (2 mL) dropwise with stirring at 0° C. The resultingsolution was stirred for 3 h at room temperature. The resulting mixturewas washed with KHSO₄ (2×10 mL). The mixture was dried over anhydroussodium sulfate and concentrated under vacuum. This resulted in 240 mg(92%) of 1-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)-3-(3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-3-oxopropyl)ureaas a yellow solid.

Intermediate 40.4:1-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)-3-(3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)propyl)urea

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of1-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)-3-(3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)-3-oxopropyl)urea(150 mg, 0.27 mmol, 1.00 equiv) in dichloromethane (10 mL) and aceticacid (1 mL) Sodium borohydride (42 mg, 1.11 mmol, 4.04 equiv) was addedand the resulting solution was stirred overnight at room temperature.The resulting mixture was washed with saturated aqueous sodium chloride(3×10 mL). The mixture was dried over anhydrous sodium sulfate andconcentrated under vacuum. This resulted in 30 mg (21%) of1-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)-3-(3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)propyl)ureaas a yellow solid.

Compound 40:2-(3-(3-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureido)propyl)malonicacid

Into a 50-mL round-bottom flask, was placed a solution of1-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)-3-(3-(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-yl)propyl)urea(100 mg, 0.19 mmol, 1.00 equiv) in 2,2,2-trifluoroacetic acid (10 mL),and water (2 mL). The resulting solution was stirred overnight at roomtemperature. The resulting mixture was concentrated under vacuum. Theresidue was applied onto a silica gel column with methanol:water (6(0?).The residue was lyophilized. This resulted in 36.3 mg (30%) of a TFAsalt of the title compound as a white solid. ¹H-NMR (300 MHz, DMSO,ppm): δ 8.55 (s, 1H), 7.64 (s, 1H), 7.39-7.42 (d, J=8.7 Hz, 2H),7.09-7.12 (d, J=8.4 Hz, 2H), 6.79 (s, 1H), 6.23-6.27 (m, 1H), 4.33-4.50(m, 3H), 3.62 (s, 1H), 3.19 (m, 1H), 3.08-3.10 (d, J=5.7 Hz, 2H), 2.94(s, 3H), 1.70-1.77 (d, J=23.1 Hz, 2H), 1.41-1.46 (d, J=12 Hz, 2H). MS(ES, m/z): 494 [M+H]⁺.

Example 41N,N′-(butane-1,4-diyl)bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Intermediate 41.1 (E)-ethyl 2-methyl-3-(3,4,5-trifluorophenyl)acrylate

To a solution of dry DMF (50 mL) under N2 was added3,4,5-trifluorobenzaldehyde (4.26 g, 26.6 mmol) followed by ethyl2-(triphenylphosphoranylidene)propionate (10.6 g, 29.3 mmol) inportions, keeping the solution at room temperature. After 1 hour, TLC(10% EtOAC in Hexanes) showed complete conversion, and the solvent wasremoved by rotary evaporation. The resulting material was brought up in50 mL methyl t-butyl ether (MBTE) and the precipitate removed byfiltration and washed with additional MBTE (3×50 mL). Afterconcentration, the resulting filtrate was applied onto a silica gelcolumn (25% EtOAc in hexanes) resulting in 6.0 g of the title compound(93%) as a white powder.

Intermediate 41.2 (E)-ethyl3-(3,5-difluoro-4-phenoxyphenyl)-2-methylacrylate

To a solution of (E)-ethyl 2-methyl-3-(3,4,5-trifluorophenyl)acrylate(Intermediate 41.1, 6.0 g, 24.56 mmol) in dry DMF (25 mL) under N2 wasadded phenol (2.774 g, 29.5 mmol) and K₂CO₃ (10.2 g, 73.68 mmol). Theresulting solution was brought to 120° C. and stirred for 3 hours atwhich point TLC indicated complete conversion. The solvent was removedby rotary evaporation and the resulting residue brought up in EtOAc (200mL) and washed with water (2×200 mL), 1N NaOH (2×200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ and concentrated to yield6.94 g (89%) of the title compound as tan crystals.

Intermediate 41.3 (E)-ethyl3-(4-(4-(chlorosulfonyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate

To a solution of (E)-ethyl3-(3,5-difluoro-4-phenoxyphenyl)-2-methylacrylate (intermediate 41.2) (1g, 3.14 mmol) in DCM (3.14 mL) under N₂ was added chlorosulfonic acid(0.419 mL, 6.28 mmol) dropwise. After 1 hour an additional 0.209 mLchlorosulfonic acid was added. After an additional hour the reactionmixture was quenched with ice-water and extracted into EtOAc (2×200 mL).The combined organic layers were dried briefly (<10 min) over Na₂SO₄ andconcentrated to recover 1.283 g of the title compound (98%) as a yellowoil.

Intermediate 41.4N,N′-(butane-1,4-diyl)bis[4-(2,6-difluoro-4-(2-carboethoxypropenyl)phenoxy)benzenesulfonamide]

To a solution of (E)-ethyl3-(4-(4-(chlorosulfonyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(Intermediate 41.3) (104.3 mg, 0.25 mmol) in chloroform (0.5 mL) wasadded DIEA (0.0869 mL, 0.5 mmol) and a solution of butane-1,4-diamine(12.6 uL, 0.125 mmol) and DIEA (0.087 mL, 0.5 mmol) in chloroform (0.125mL). After one hour the solvent was removed and the resulting residuebrought up in EtOAc (40 mL), washed with water (2×40 mL), brine (40 mL)and dried over Na₂SO₄. Removing the solvent gave 118 mg of the titlecompound which was used without further purification.

Intermediate 41.5:N,N′-(butane-1,4-diyl)bis[4-(2,6-difluoro-4-(2-carboxypropenyl)phenoxy)benzenesulfonamide]

To a solution of Intermediate 41.4 (118 mg, 0.139 mmol) in MeOH (1.39mL) was added a NaOH (0.3M in water, 0.278 mL, 0.835 mmol). The reactionwas placed under N₂ and heated at 60° C. for 30 minutes. After coolingthe reaction mixture was diluted with water (20 mL), partitioned withEtOAc (20 mL) and acidified with HCl. After extracting with EtOAc (2×20mL) the combined organic phases were dried over Na₂SO₄ and the solventremoved to give 40.7 mg of the title compound.

Compound 41:N,N′-(butane-1,4-diyl)bis[(E)-N-(diaminomethylene)₃-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Thionyl chloride (2 mL) was added to intermediate 41.5 (40.7 mg, 0.051mmol) and was heated at 80° under N₂. After 70 minutes, the solvent wasremoved in vacuo. The residue was brought up in toluene (2 mL) and thetoluene was also removed in vacuo. The bis-acid chloride was dissolvedin DME (0.5 mL) and added to guanidine free base (1.4 mmol, prepared asfollows: To a slurry of guanidine hydrochloride (480 mg, 5.0 mmol) wasadded 25% NaOMe in MeOH (1.03 mL, 4.5 mmol). The mixture was stirred for30 minutes and then filtered. A portion of the filtrate (0.40 mL) wasconcentrated to dryness.) in DME (1 mL). After 15 minutes, water (10 mL)was added and the mixture was extracted with EtOAc (3×25 mL). Theorganic layer was dried (Na₂SO₄) and concentrated. The crude product waspurified by preparative HPLC to give the title compound (7.8 mg) as theTFA salt. ¹H-NMR (400 mHz. CD₃OD) δ 7.80 (d, 4H), 7.44 (s, 2H), 7.30 (d,4H), 7.11 (d, 4H), 2.80 (m, 4H), 2.18 (s, 6H), 1.44 (m, 4H). MS (m/z):875.16 (M+H).

Example 42N,N′-(1,4-phenylenebis(methylene))bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Compound 42:N,N′-(1,4-phenylenebis(methylene))bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide])

Following the procedures outlined in Example 41, compound 42 was madeusing 1,4-phenylenedimethanamine as the amine. Purification bypreparative HPLC gave the title compound as a TFA salt. ¹H-NMR (400 mHz.CD3OD) δ 7.87 (di, 4H), 7.44 (s, 2H), 7.31 (d, 4H), 7.06 (d, 6H), 7.04(s, 2H), 4.02 (s, 4H), 2.19 (s, 6H). MS (m/z): 924.21 (M+H)

Example 43N,N′-(2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis[(E)-N-(diaminomethylene)(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Intermediate 43.1N,N′-(2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis((E)-4-(2,6-difluoro-4-(2-carboethoxypropenyl)phenoxy)benzenesulfonamide)

To a solution of (E)-ethyl3-(4-(4-(chlorosulfonyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(intermediate41.3) (225 mg, 0.54 mmol) in DCM (3 mL) was added a solution of2,2′-(ethane-1,2-diylbis(oxy))diethanamine (38 mg, 0.26 mmol) andtriethylamine (101 mg, 1.0 mmol) in DCM (2 mL) dropwise. After 30minutes, 1N HCl was added (10 mL) and the reaction mixture was extractedwith DCM (3×15 mL). The combined organic layers were dried (Na₂SO₄) andconcentrated to give the title compound (262 mg).

Intermediate 43.2N,N′-(2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis((E)-4-(2,6-difluoro-4-(2-carboxypropenyl)phenoxy)benzenesulfonamide)

A solution of the intermediate 43.1 (262 mg, 0.29 mmol) and 3N NaOH (0.6mL, 1.8 mmol) in methanol (3 mL) was heated at 65° C. for 1 hour. Thereaction mixture was cooled to RT and the methanol removed at reducedpressure and 1N HCl (3 mL, 3 mmol) was added to the residue. The productwas extracted into DCM (3×15 mL). The combined organic layers were dried(Na₂SO₄) and concentrated to give the title compound (173 mg).

Compound 43:N,N′-(2,2′-(ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Thionyl chloride (1 mL) was added to intermediate 43.2 (63 mg, 0.074mmol) and was heated at 80°. After 2 hours, the solvent was removed invacuo. The bis-acid chloride was dissolved in DME (1 mL) and added toguanidine free base (1.4 mmol, prepared as follows: To a slurry ofguanidine hydrochloride (480 mg, 5.0 mmol) was added 25% NaOMe in MeOH(1.03 mL, 4.5 mmol). The mixture was stirred for 30 minutes and thenfiltered. A portion of the filtrate (0.40 mL) was concentrated todryness.) in DME (1 mL). After 15 minutes, water (10 mL) was added andthe mixture was extracted with EtOAc (3×25 mL). The organic layer wasdried (Na₂SO₄) and concentrated. The crude product was purified bypreparative HPLC to give the title compound (20 mg) as the TFA salt.¹H-NMR (400 mHz, CD₃OD) δ 7.83 (d, j=8.8 Hz, 4H), 7.43 (s, 2H), 7.30 (d,j=8.9 Hz, 4H), 7.11 (d, j=8.6 Hz, 4H), 3.42 (t, j=5.5 Hz, 8H), 3.03 (t,j=5.4 Hz, 4H), 2.17 (s, 6H). MS (m/z): 935.08 (M+H).

Example 44N,N′-(2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethane-2,1-diyl))bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Intermediate 44.1: (E)-ethyl3-(4-(4-(N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate

To a solution of (E)-ethyl3-(4-(4-(chlorosulfonyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(intermediate 41.3) (250 mg, 0.60 mmol) in DCM (3 mL) was added asolution of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (157 mg,0.72 mmol) and triethylamine (72 mg, 0.72 mmol) in DCM (2 mL). After 15minutes, water (10 mL) was added and the reaction mixture was extractedwith DCM (2×25 mL). The combined organic layers were washed with water(10 mL), brine (10 mL), dried (Na₂SO₄) and concentrated. The crudematerial was purified by flash chromatography on silica gel eluting with50% EtOAc in DCM to give the title compound (169 mg).

Intermediate 44.2: (E)-ethyl3-(4-(4-(N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate

To a solution of (E)-ethyl3-(4-(4-(N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(169 mg, 0.28 mmol) in THF (6 ml) and water (0.6 mL) under nitrogen wasadded trimethylphosphine (26 mg, 0.34 mmol). After stirring for 3 hours,the solvents were removed at reduced pressure and. The residue wasdissolved in water (5 mL) and extracted with EtOAc (3×25 mL). Thecombined organic layers were dried (Na₂SO₄) and concentrated to give thetitle compound (162 mg).

Intermediate 443:N,N′-(2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethane-2,1-diyl))bis[4-(2,6-difluoro-4-(2-carboethoxypropenyl)phenoxy)benzenesulfonamide]

A solution of (E)-ethyl3-(4-(4-(chlorosulfonyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(intermediate 41.3) (71 mg, 0.17 mmol) in EtOAc (1 mL) was added to asolution of (E)-ethyl3-(4-(4-(N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(84 mg, 0.15 mmol) and triethylamine (22 mg, 0.22 mmol) in DCM (1 mL)with stirring. After 30 minutes, water (10 mL) was added and the productextracted into DCM (3×15 mL). The combined organic layers were dried(Na₂SO₄) and concentrated to give the title compound (177 mg).

Compound 44N,N′-(2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethane-2,1-diyl))bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Following the procedures outlined in Example 43, intermediate 44.3 wasconverted to the bis-guanidine and gave, after purification bypreparative HPLC, the title compound (21 mg) as a TFA salt. ¹H-NMR (400mHz, CD3OD) δ 7.84 (d, j=8.8 Hz, 4H), 7.44 (s, 2H), 7.30 (d, j=8.8 Hz,4H), 7.10 (d, j=8.8 Hz, 4H), 3.54 (m, 4H), 3.48 (m, 4H), 3.43 (t, j=5.5Hz, 4H), 3.04 (t, j=5.5 Hz, 4H), 2.17 (d, j=1.2 Hz, 6H). MS (m/z):979.05 (M+H).

Example 45(E)-3-(4-(4-(N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-N-(diaminomethylene)-2-methylacrylamide

Compound 45:(E)-34-4(4-(N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-N-(diaminomethylene)-2-methylacrylamide

A 4.3 M solution of guanidine free base in methanol was prepared. A 25%solution of NaOMe in MeOH (1.03 mL, 4.5 mmol) was added to guanidinehydrochloride (480 mg, 5.0 mmol), and the mixture was stirred for 30minutes. The mixture was filtered (0.2μ, PTFE) to give the guanidinefree base solution. A portion (0.3 mL, 1.3 mmol) was added to (E)-ethyl3-(4-(4-(N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(74 mg, 0.13 mmol) with stirring. After 15 minutes, water (10 mL) wasadded and the product extracted with DCM (4×20 mL). The combined organiclayers were dried (Na₂SO₄) and concentrated. The crude product waspurified by preparative HPLC to give the title compound (34 mg) as a TFAsalt. ¹H-NMR (400 mHz, d6-DMSO) δ 11.14 (s, 1H), 8.38 (br s, 4H), 7.78(d, j=9.0 Hz, 2H), 7.5 (m, 3H), 7.45 (d, j=9.1, 2H), 7.42 (s, 1H), 7.19(d, j=8.8 Hz, 2H), 3.55 (m, 6H), 3.44 (m, 4H), 3.36 (m, 2H), 2.95 (m,2H), 2.87 (m, 2H), 2.11 (s, 3H). MS (m/z): 586.11 (M+H).

Example 46N,N′-(13-oxo-3,6,9,17,20,23-hexaoxa-12,14-diazapentacosane-1,25-diyl)bis[(E)-N-(diaminomethylene)-3(3,5-difluoro-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Intermediate 46.1N,N′-(13-oxo-3,6,9,17,20,23-hexaoxa-12,14-diazapentacosane-1,25-diyl)bis[4-(2,6-difluoro-4-(2-carboethoxypropenyl)phenoxy)benzenesulfonamide]

Carbonyldiimidisole (16.2 mg, 0.10 mmol) was added to a solution of(E)-ethyl3-(4-(4-(N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(intermediate 44.2) (125 mg, 0.22 mmol) in DMF (2 mL) and stirred for 23hours at which time the solvent was removed under vacuum. The residuewas dissolved in EtOAc, washed with water (4×10 mL), dried (Na₂SO₄) andconcentrated to give the title compound (132 mg).

Compound 46:N,N′-(13-oxo-3,6,9,17,20,23-hexaoxa-12,14-diazapentacosane-1,25-diyl)bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

A solution of 4.4 M guanidine in methanol (Example 45) (0.5 mL, 2.2mmol) was added to a solution of intermediate 46.1 (65 mg, 0.055 mmol)in DMF, and stirred for 4 hours. The reaction was quenched with 50%aqueous AcOH, and then concentrated to dryness. The residue was purifiedby preparative HPLC to give the title compound (35 mg) as a TFA salt.¹H-NMR (400 mHz, CD3OD) δ 7.84 (d, j=8.2 Hz, 4H), 7.43 (d, j=1.4 Hz,2H), 7.30 (d, j=9.0 Hz, 4H), 7.11 (d, j=−9.0 Hz, 4H), 3.57 (m, 12H),3.46 (m, 12H), 3.26 (t, J=5.4 Hz, 4H), 3.04 (t, j=5.4 Hz, 4H), 2.17 (d,j=1.3 Hz, 6H). MS (m/z): 1197.07 (M+H).

Example 47 N,N′-(13,20 dioxo-3, 6, 9, 24, 27, 30-hexaoxa-12,21-diazadotriacontane-1,32-diyl)bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Compound 47: N,N′-(13,20 dioxo-3, 6, 9, 24, 27, 30-hexaoxa-12,21-diazadotricontane-1,32-diyl)bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Following the procedures in Example 46, substituting subaric acidbis(N-hydroxysuccinimide ester) for carbonyldiimidazole gave the titlecompound as a TFA salt. ¹H-NMR (400 mHz, CD3OD) δ 7.84 (m, 4H), 7.43 (m,2H), 7.30 (m, 4H), 7.11 (m, 4H), 3.58 (m, 12H), 3.50 (m, 8H), 3.32 (m,4H), 3.05 (t, j=5.4 Hz, 4H), 2.18 (d, j=1.6 Hz, 6H), 2.15 (m, 4H), 1.56(m, 4H), 1.29 (m, 4H). MS (m/z): 1309.12 (M+H).

Example 48(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-(N-(2-(2-(2-(2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)phenyl)-2-methylacrylamide

Intermediate 48.1:(E)-3-(4-(4-(N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-N-(diaminomethylene)-2-methylacrylamide

To (E)-ethyl3-(4-(4-(N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(250 mg, 0.42 mmol) was added 4.4 M guanidine in in methanol (asprepared in example 45) (1.0 mL, 4.4 mmol) and the reaction was stirredat RT. After 30 minutes, water (10 mL) was added, and the mixture wasextracted with DCM (4×25 mL). The aqueous phase was adjusted to pH 7,and extracted with DCM (2×25 mL). The combined organic extracts weredried (Na₂SO₄) and concentrated to give the title compound (245 mg).

Compound 48:(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-(N-(2-(2-(2-(2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)phenyl)-2-methylacrylamide

To a mixture of(E)-3-(4-(4-(N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-N-(diaminomethylene)-2-methylacrylamide(70 mg, 0.11 mmol) and propargyl alcohol (6.4 mg, 0.11 mmol) int-butanol (0.22 mL) and water (0.22 mL) was added 1 M sodium ascorbate(11 μL, 0.011 mmol) and 0.3 M copper sulfate (3.6 μL, 0.0011 mmol) andthe reaction was stirred at RT. After 14 hours, the product was purifiedby preparative HPLC to give the title compound (22 mg) as a TFA salt.¹H-NMR (400 mHz, CD₃OD) δ 7.93 (s, 1H), 7.84 (m, 2H), 7.44 (s, 1H), 7.30(m, 2H), 7.11 (m, 2H), 4.64 (d, j=0.6 Hz, 2H), 4.55 (t, j=5.0 Hz, 2H),3.86 (t, j=5.0 Hz, 2H), 3.57 (m, 4H), 3.52-3.42 (m, 6H), 3.03 (t, j=5.4Hz, 2H), 2.18 (d, j=1.3 Hz, 3H). MS (m/z): 668.14 (M+H).

Example 49N,N′-(2,2′-(2,2′-(2,2′-(2,2′-(4,4′-oxybis(methylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Compound 49:N,N′-(2,2′-(2,2′-(2,2′-(2,2′-(4,4′-oxybis(methylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis[(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide]

Following the procedures in example 48, substituting propargyl ether forpropargyl alcohol gave the title compound as a TFA salt. ¹H-NMR (400mHz, CD3OD) δ 8.00 (s, 2H), 7.83 (m, 4H), 7.43 (s, 2H), 7.30 (m, 4H),7.10 (m, 4H), 4.61 (s, 4H), 4.55 (m, 4H), 3.86 (m, 4H), 3.58-3.50 (m,8H), 3.50-3.40 (m, 12H), 3.01 (m, 4H), 2.17 (d, j=1.3 Hz, 6H). MS (m/z):1317.09 (M+H).

Example 50N,N′-(2,2′-(piperazine-1,4-diyl)bis(ethane-2,1-diyl))di-((E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide)

Intermediate 50.1: 2,2′-(piperazine-1,4-diyl)diacetonitrile

To a solution of piperazine (6 g, 69.77 mmol, 1.00 equiv) inacetonitrile (150 mL) was added potassium carbonate (19.2 g, 139.13mmol, 2.00 equiv) and the mixture was stirred. To this was addeddropwise a solution of 2-bromoacetonitrile (16.7 g, 140.34 mmol, 2.00equiv) in acetonitrile (100 mL) and the suspension was stirred for 4 hat room temperature. The solids were filtered out and the resultingsolution was concentrated under vacuum. The crude product was purifiedby re-crystallization from methanol resulting in 7.75 g (68%) ofIntermediate 50.1 as a white solid.

Intermediate 50.2: 2,2′-(piperazine-1,4-diyl)diethanamine

To a suspension of lithium aluminum hydride (LiAlH₄; 700 mg, 18.42 mmol,4.30 equiv) in tetrahydrofuran (40 mL) cooled to 0° C. was addeddropwise a solution of Intermediate 50.1 (700 mg, 4.27 mmol, 1.00 equiv)in tetrahydrofuran (10 mL). The mixture was stirred for 15 minutes at 0°C. and heated to reflux for 3 h. The reaction was cooled, the pHadjusted to 8-9 with potassium hydroxide (50%), and the solids filteredout. The resulting mixture was concentrated under vacuum and theresulting solids washed with hexane to afford 0.3 g (416%) ofIntermediate 50.2 as a yellow solid.

Intermediate 503:N,N′-(2,2′-(piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(4-(benzyloxy)benzenesulfonamide)

To Intermediate 50.2 (500 mg, 2.91 mmol, 1.00 equiv) in dichloromethane(10 mL) was added triethylamine (1.46 g, 0.01 mmol, 2.00 equiv) and4-(benzyloxy)benzene-1-sulfonyl chloride (2.0 g, 0.01 mmol, 2.40 equiv)and the resulting solution was stirred for 2 h at room temperature. Thereaction was diluted with dichloromethane, washed with 3×10 mL of water,dried over sodium sulfate then filtered and concentrated under vacuum toafford 0.9 g (47%) of Intermediate 50.3 as a yellow solid.

Intermediate 50.4:N,N′-(2,2′-(piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(4-hydroxybenzenesulfonamide)

To intermediate 50.3 (3 g, 4.52 mmol, 1.00 equiv) inN,N-dimethylformamide (500 mL) and methanol (100 mL) was added Palladiumon carbon (1 g) and the suspension stirred under hydrogen gas for 4 h atroom temperature. The solids were filtered out and the resulting mixturewas concentrated under vacuum to afford 1.5 g (69%) of Intermediate 50.4as a gray solid.

Intermediate 50.5:N,N′-(2,2′-(piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis((E)-ethyl3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylate)

To Intermediate 50.4 (1 g, 2.06 mmol, 1.00 equiv) inN,N-dimethylformamide (30 mL) was added Cs₂CO₃ (1.45 g, 4.45 mmol, 2.16equiv) and the resulting suspension stirred for 2 h at room temperature.To this was added a solution of (E)-ethyl2-methyl-3-(3,4,5-trifluorophenyl)acrylate (intermediate 41.1) (1.1 g,4.51 mmol, 2.19 equiv) in N,N-dimethylformamide (10 mL) dropwise withstirring. The reaction was stirred for 0.5 h at room temperature andthen overnight at 90° C. The resulting mixture was concentrated undervacuum, the residue was applied onto a silica gel column and then elutedwith dichloromethane:methanol (100:1) to afford 390 mg (20%) ofIntermediate 50.5 as a yellow solid.

Intermediate 50.6:N,N′-(2,2′-(piperazine-1,4-diyl)bis(ethane-2,1-diyl))di-((E)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylicacid)

To Intermediate 50.5 (170 mg, 0.16 mmol, 1.00 equiv, 90%) in 1:1methanol/tetrahydrofuran (20 mL) was added lithium hydroxide (4 equiv,30 mg) and the reaction was stirred for 2 h at 27° C. The pH value ofthe solution was adjusted to 1-2 with aqueous hydrochloric acid (6mol/L) and the solids were collected by filtration. The residue waswashed with ethyl acetate (2×5 mL) and then dried under vacuum to afford150 mg (94%) of Intermediate 50.6 as a white solid.

Compound 50:N,N′-(2,2′-(piperazine-1,4-diyl)bis(ethane-2,1-diyl))di-((E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylamide)

To a solution of Intermediate 50.6 (100 mg, 0.09 mmol, 1.00 equiv, 80%)in tetrahydrofuran (30 mL) was added carbonyl diimidazole (CDI; 58 mg,0.36 mmol, 4.00 equiv) and the resulting solution was stirred for 1 h at25° C. To this was added guanidine (2M in methanol, 10 ml) and theresulting solution was stirred for an additional 14 h at 30° C. Theresulting mixture was concentrated under vacuum, the residue was appliedonto a silica gel column and eluted with dichloromethane:methanol(10:1). The crude product (230 mg) was then purified by reverse-phase(C18) preparative-HPLC to afford 16 mg (17%) of a formate salt of thetitle compound as a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): 7.89-7.92(4H, d, J=8.7 Hz), 7.50 (2H, s), 7.34-7.36 (4H, d, J=8.7 Hz), 7.16-7.19(4H, d, J=8.7 Hz), 2.88-3.16 (16H, m), 2.20 (6H, s); MS (ES, m/z): 959[M+H]⁺

Example 51(E)-4-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)phenylphosphonicacid

Intermediate 51.1: (E)-3-(3,5-difluoro-4-phenoxyphenyl)-2-methylacrylicacid

To a solution of (E)-ethyl3-(3,5-difluoro-4-phenoxyphenyl)-2-methylacrylate (intermediate 41.2)(900 mg, 2.83 mmol, 1.00 equiv) in methanol (20 mL) was added methanolic2M LiOH (50 mL) and the resulting solution stirred for 2 h. Theresulting mixture was concentrated under vacuum, the pH value of thesolution was adjusted to 5-6 with aqueous HCl (6 mol/L) and the mixturewas extracted with 3×20 mL of ethyl acetate. The organic layers werecombined, washed with 2×10 mL of sodium chloride (sat.) and then driedover anhydrous sodium sulfate. The solids were filtered out and thesolution was concentrated to afford 0.7 g (85%) of Intermediate 51.1 asa white solid.

Intermediate 51.2:(E)-3-(4-(4-(chlorosulfonyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylicacid

To Intermediate 51.1 (1 g, 3.14 mmol, 1.00 equiv) in dichloromethane (15mL) at 0-5° C. was added dropwise a solution of sulfurochloridic acid(8.5 g, 73.28 mmol, 23.00 equiv) in dichloromethane (5 mL). The reactionwas stirred overnight at 25° C. in an oil bath, and then quenched by theaddition of 200 mL of water/ice. The mixture was extracted with 4×50 mLof dichloromethane and the organic layers combined and dried overanhydrous sodium sulfate to afford 1.1 g (90%) of Intermediate 51.2 as ayellow solid.

Intermediate 51.3:(E)-3-(4-(4-(N-(4-(diethoxyphosphoryl)phenyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylicacid

To diethyl 4-aminophenylphosphonate (intermediate 2.2) (150 mg, 0.66mmol, 1.00 equiv) in pyridine (3 mL) was added Intermediate 51.2 (300mg, 0.77 mmol, 1.22 equiv) in several portions. The mixture was stirredfor 3 h at 30° C. and then concentrated, the pH value of the solutionadjusted to 3 with aqueous HCl (1 mol/L) and the resulting mixtureextracted with 3×30 mL of ethyl acetate. The organic layers werecombined, dried over anhydrous sodium sulfate, concentrated, appliedonto a silica gel column and eluted with dichloromethane:methanol (50:1)to afford 100 mg (26%) of Intermediate 51.3 as a yellowish solid.

Intermediate 51.4: (E)-diethyl4-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)phenylphosphonate

To Intermediate 51.3 (150 mg, 0.26 mmol, 1.00 equiv) in tetrahydrofuran(2 mL) was added CDI (120 mg, 0.74 mmol, 1.40 equiv) and the reactionstirred for 2 h at RT. To this was added guanidine (1M in DMF; 0.8 ml)and the reaction was stirred overnight at 30° C. The resulting mixturewas concentrated under vacuum and the crude product was purified byreverse phase (C18) Prep-HPLC to afford 40 mg (25%) of Intermediate 51.4as a White solid.

Compound 51:(E)-4-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)phenylphosphonicacid

To Intermediate 51.4 (40 mg, 0.06 mmol, 1.00 equiv) in tetrahydrofuran(2 mL) was added bromotrimethylsilane (15 mg, 0.09 mmol, 1.37 equiv)dropwise with stirring and the resulting solution was stirred at 40° C.overnight. The resulting mixture was concentrated, diluted with methanol(2 mL) and then concentrated under vacuum. This operation was repeatedfour times. The crude product (75 mg) was purified by reverse phase(C18) Prep-HPLC to afford 12.5 mg of a formate salt of the titlecompound as a white solid. ¹H-NMR (300 MHz, DMSO, ppm): 10.54 (s, 1H),7.82-7.79 (d, J=8.4 Hz, 2H), 7.52-7.40 (m, 5H), 7.18-7.10 (m, 4H), 2.08(s, 3H): ³¹P-NMR (400 MHz, DMSO, ppm): 11.29; MS (ES, m/z): 567 [M+H]⁺

Example 52(E)-4-((4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)methyl)benzylphosphonicacid

Intermediate 52.1: diethyl4-((4-(benzyloxy)phenylsulfonamido)methyl)benzyl-phosphonate

To 4-diethyl 4-(aminomethyl)benzylphosphonate (intermediate 6.1) (60 mg,0.23 mmol, 1.00 equiv) in dichloromethane (10 mL), triethylamine (47 mg,0.47 mmol, 2.00 equiv) was added dropwise a solution of4-(benzyloxy)benzene-1-sulfonyl chloride (72 mg, 0.26 mmol, 1.10 equiv)in dichloromethane (5 mL) and the resulting solution was stirred for 1 hat 25° C. The reaction mixture was concentrated, the residue appliedonto a silica gel column and then eluted with ethyl acetate/petroleumether (1:1). The isolated product was washed with 2×50 mL of n-hexaneresulting in 50 mg (43%) of Intermediate 52.1 as a white solid.

Intermediate 52.2: diethyl4-((4-hydroxyphenylsulfonamido)methyl)benzyl-phosphonate

To Intermediate 52.1 (1.2 g, 2.39 mmol, 1.00 equiv) in methanol (20 mL)in N,N-dimethylformamide (5 mL) was added Palladium on carbon (0.9 g)and the suspension stirred overnight at 30° C. under a hydrogenatmosphere. The reaction was filtered and concentrated under vacuum toafford 1 g (91%) of Intermediate 52.2 as brown oil.

Intermediate 52.3: (E)-ethyl-(4-(4-(N-(4-((diethoxyphosphoryl)methyl)benzyl)-sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate

To Intermediate 52.2 (100 mg, 0.24 mmol, 1.00 equiv) inN,N-dimethylformamide (10 mL) was added Cs₂CO₃ (160 mg, 0.49 mmol, 2.10equiv) and the mixture was stirred for 1.5 h at room temperature. Tothis was added a solution of (E)-ethyl2-methyl-3-(3,4,5-trifluorophenyl)acrylate (intermediate 41.1) (60 mg,0.25 mmol, 1.10 equiv) in N,N-dimethylformamide (5 mL) and the reactionwas stirred overnight at 90° C. The solids were filtered out and thefiltrate was concentrated under vacuum, the residue applied onto asilica gel column and eluted with dichloromethane/methanol (200:1) toafford 50 mg (23%) of Intermediate 52.3 as yellow oil.

Intermediate 52.4:(E)-3-(4-(4-(N-(4-((diethoxyphosphoryl)methyl)benzyl)sulfamoyl)-phenoxy)-3,5-difluorophenyl)-2-methylacrylicacid

To Intermediate 52.3 (700 mg, 1.10 mmol, 1.00 equiv) in tetrahydrofuran(20 mL) and water (20 mL) was added LiOH (700 mg, 29.17 mmol, 30.00equiv) and the resulting solution was stirred for 1 h at 25° C. Thereaction was concentrated, the pH value of the solution was adjusted to4-5 with aqueous HCl (2 mol/L) and the mixture was extracted with 2×150mL of ethyl acetate. The organic layers were combined, dried overanhydrous sodium sulfate, concentrated, the residue applied onto asilica gel column and then eluted with ethyl acetate/petroleum ether(1:1-2:1) to afford 250 mg (35%) of Intermediate 52.4 as a white solid.

Compound 52:(E)-4-((4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)methyl)benzylphosphonicacid

Compound 52 was prepared from Intermediate 52.4 using the proceduresdescribed under Example 51, except preparative HPLC was not required,affording 84 mg (89%) of a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm):7.83-7.80 (d, J=8.7 Hz, 2H), 7.52 (s, 1H), 7.38-7.36 (d, J=8.7 Hz, 2H),7.23-7.20 (m, 2H), 7.17-7.09 (m, 4H), 4.06 (s, 2H), 3.11 (s, 1H), 3.04(s, 1H), 2.23-2.23 (s, 3H). MS (ES, m/z): 595 [M+H]⁺.

Example 53(E)-4-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)benzylphosphonicacid

Compound 53:(E)-4-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)benzylphosphonicacid

Compound 53 was prepared from diethyl 4-aminobenzylphosphonate(intermediate 3.2) using the procedures described in Example 52 exceptthe final product was purified by preparative HPLC. ¹H-NMR (300 MHz,CD₃OD, ppm): 7.77-7.74 (d, J=8.7 Hz, 2H), 7.46 (s, 1H), 7.33-7.31 (d,J=8.7 Hz, 2H), 7.21-7.19 (m, 2H), 7.06-7.11 (m, 4H), 3.04-2.97 (d,J=21.6 Hz, 2H), 2.19 (s, 3H); ³¹P-NMR (400 MHz, CD₃OD, ppm): 22.49. MS(ES, m/z): 581 [M+H]⁺.

Example 54(E)-3-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)propylphosphonicacid

Compound 54:(E)-3-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)propylphosphonicacid

Compound 54 was prepared from diethyl 3-aminopropylphosphonate(intermediate 4.1) using the procedures described under Example 51.¹H-NMR (400 MHz, DMSO, ppm): 7.81-7.78 (d, J=8.4 Hz, 2H), 7.57 (s, 1H),7.42-7.39 (d, J=9.3 Hz, 2H), 7.22-7.19 (d, J=8.7 Hz, 2H), 2.75-2.77 (q,2H), 2.10 (s, 3H), 1.59-1.42 (m, 4H). MS (ES, m/z): 533 [M+H]⁺

Example 55(E)-2-((3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)ethylphosphonicacid

Compound 55:(E)-2-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)ethylphosphonicacid

Compound 55 was prepared from diethyl 2-aminoethylphosphonate(intermediate 1.9) using the procedures described under Example 51,except purification of the final product by preparative HPLC was notrequired. ¹H-NMR (400 MHz, DMSO, ppm): 11.02 (s, 1H), 8.28 (s, 4H),7.79-7.82 (d, J=9.2 Hz, 2H), 7.62-7.65 (t, 1H), 7.54-7.49 (m, 3H),7.26-7.24 (d, J=8.8 Hz, 2H), 3.42-3.58 (m, 2H), 2.15 (s, 3H), 1.73-1.65(m, 2H); ³¹P-NMR (400 MHz, DMSO, ppm): 21.36. MS (ES, m/z): 519 [M+H]⁺

Example 56(E)-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)methylphosphonicacid

Compound 56:(E)-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)methylphosphonicacid

Compound 56 was prepared from diethyl aminomethylphosphonate(intermediate 5.3) using the procedures described under Example 51,except purification of the final product by Flash-Prep-HPLC withCH₃CN:water (10:100). ¹H -NMR (300 MHz, DMSO, ppm): δ 7.84-7.81 (d,J=8.1 Hz, 2H), 7.57 (s, 1H), 7.45-7.42 (d, J=9.3 Hz, 3H), 7.18-7.15 (d,J=8.4 Hz, 2H), 3.04-3.01 (m, 2H), 2.08 (s, 3H). MS (ES, m/z): 505[M+H]⁺.

Example 57(E)-2-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)-N-(phosphonomethyl)phenylsulfonamido)aceticacid

Compound 57:(E)-2-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)-N-(phosphonomethyl)phenyl-sulfonamido)aceticacid

Compound 57 was prepared from ethyl2-((diethoxyphosphoryl)methylamino)acetate (intermediate 8.2) using theprocedures described under Example 51. ¹H-NMR (300 MHz, DMSO, ppm): δ8.33 (s, 4H), 7.84-7.81 (d, J=8.1 Hz, 2H), 7.52-7.50 (d, J=7.8 Hz, 2H),7.19-7.16 (d, J=8.4 Hz, 2H), 4.11 (s, 2H), 2.14 (s, 3H); MS (ES, m/z):563 [M+H]⁺.

Example 58(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-(N-(2-methoxyethylcarbamoyl)sulfamoyl)phenoxy)phenyl)-2-methylacrylamide

Intermediate 58.1:(E)-3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylic acid

(E)-3-(4-(4-(chlorosulfonyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylicacid (Intermediate 51.2) was converted to intermediate 58.1 usingprocedures outlined in Example 58, with aqueous ammonia as the amine.The title compound was obtained as a yellow solid.

Intermediate 58.2: (E)-methyl3-(3,5-difluoro-4-(4-sulfamoylphenoxy)phenyl)-2-methylacrylate

Into a 50-mL round-bottom flask, was placed a solution of intermediate58.1 (2 g, 5.42 mmol, 1.00 equiv) in methanol (60 mL). This was followedby the addition of thionyl chloride (2.5 g, 21.19 mmol, 4.00 equiv)dropwise with stirring at 0° C. The resulting solution was stirred for 3h at 50° C. The resulting mixture was concentrated under vacuum. The pHvalue of the solution was adjusted to 7 with ammonia (2 mol/L). Theresulting solution was extracted with 10 mL of ethyl acetate and theorganic layers combined and dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was applied onto a silica gelcolumn with petroleum ether/ethyl acetate (30:1-1:1). This resulted in2.1 g (97%) of the title compound as a white solid.

Intermediate 58.3: (E)-methyl3-(4-(4-(N-(ethoxycarbonyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate

Into a 50-mL round-bottom flask, was placed a solution of intermediate58.2 (280 mg, 0.73 mmol, 1.00 equiv) in acetone (20 mL). This wasfollowed by the addition of potassium carbonate (200 mg, 1.45 mmol, 2.00equiv). The mixture was stirred for 3 h at room temperature. To this wasadded ethyl chloroformate (90 mg, 0.83 mmol, 1.20 equiv). The resultingsolution was stirred for 6 h at 65° C. The resulting mixture wasconcentrated under vacuum. The pH value of the solution was adjusted to2-3 with hydrogen chloride (1 mol/L). The resulting solution wasextracted with 2×50 ml of ethyl acetate and the organic layers combined.The mixture was dried over anhydrous sodium sulfate and concentratedunder vacuum. This resulted in 300 mg (72%) of the title compound asyellow oil.

Intermediate 58.4: (E)-methyl3-(3,5-difluoro-4-(4-(N-(2-methoxyethylcarbamoyl)-sulfamoyl)phenyl)-2-methylacrylate

Into a 100-mL round-bottom flask, was placed a solution of intermediate58.3 (300 mg, 0.66 mmol, 1.00 equiv) in toluene (20 mL),2-methoxyethanamine (100 mg, 1.33 mmol, 1.10 equiv). The resultingsolution was stirred for 1 h at 110° C. The resulting mixture wasconcentrated under vacuum. The residue was applied onto a silica gelcolumn with petroleum ether/ethyl acetate (1:1). This resulted in 0.3 g(92%) of the title compound as a yellow solid.

Compound 58:(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-(N-(2-methoxyethylcarbamoyl)sulfamoyl)phenoxy)phenyl)-2-methylacrylamide

Intermediate 58.4 was converted to compound 58 using the proceduresdescribed under Example 52. Purification by preparative HPLC gave a TFAsalt of the title compound. ¹H-NMR (300 MHz, DMSO, ppm): δ10.62 (s, 1H),8.33 (s, 3H), 7.94-7.91 (d, J=8.7 Hz, 2H), 7.55-7.52 (d, J=9 Hz, 2H),7.45 (s, 1H), 7.26-7.22 (d, J=9 Hz, 2H), 6.55 (s, 1H), 3.37-3.27 (m,2H), 3.21 (s, 3H), 3.15-3.12 (m, 2H), 2.16 (s, 3H). MS (ES, m/z): 512[M+H]⁺.

Example 59(E)-2-((3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)succinicacid

Intermediate 59.1: (E)-di-tert-butyl2-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)succinate

Intermediate 59.1 was prepared from di-tert-butyl 2-aminosuccinate usingthe procedures described under Example 51.

Compound 59:(E)-2-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)succinicacid

Into a 50-mL round-bottom flask, was placed a solution of intermediate59.1 (100 mg, 0.16 mmol, 1.00 equiv) in tetrahydrofuran (5 mL). This wasfollowed by the addition of 2,2,2-trifluoroacetic acid (10 mL) dropwisewith stirring. The resulting solution was stirred for 3 h at roomtemperature. The resulting mixture was concentrated under vacuum. Thisresulted in 63.6 mg (64%) of a TFA salt of the title compound as a lightyellow solid. ¹H-NMR (300 MHz, DMSO, ppm): δ 8.26 (s, 4H), 7.82-7.79 (d,J=8.7 Hz, 2H), 7.49-7.45 (m, 3H), 7.19-7.16 (d, J=8.4 Hz, 2H), 4.00-3.96(m, 1H), 2.65-2.60 (m, 1H), 2.48-2.41 (m, 1H), 2.13 (s, 3H). MS (ES,m/z): 527 [M+H]⁺.

Example 604-(3-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazine-1-carboximidamide

Intermediate 60.1: tert-butyl 4-(3-bromophenyl)piperazine-1-carboxylate

Into a 250-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed copper(I) iodide (1.0 g, 5.26 mmol,0.20 equiv), L-proline (930 mg, 8.09 mmol, 0.30 equiv) in DMSO (50 mL).The resulting solution was stirred for 15 min at room temperature. Then,tert-butyl piperazine-1-carboxylate (5 g, 26.88 mmol, 1.00 equiv),1,3-dibromobenzene (9.5 g, 40.25 mmol, 1.50 equiv), potassium carbonate(7.4 g, 53.62 mmol, 1.99 equiv) was added. The resulting solution wasstirred overnight at 90° C. The reaction was then quenched by theaddition of 100 mL of water. The resulting solution was extracted with2×100 mL of ethyl acetate and the organic layers combined and dried overanhydrous sodium sulfate and concentrated under vacuum. The residue wasapplied onto a silica gel column with ethyl acetate/petroleum ether(1:6). This resulted in 2.9 g of tert-butyl4-(3-bromophenyl)piperazine-1-carboxylate as a white solid.

Intermediate 60.2:3-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenylboronic acid

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of tert-butyl4-(3-bromophenyl)piperazine-1-carboxylate (3.8 g, 11.14 mmol, 1.00equiv) in toluene/tetrahydrofuran=1:1 (40 mL). This was followed by theaddition of n-BuLi (4.9 mL, 2.5M/L) dropwise with stirring at −70° C.The resulting solution was stirred for 30 min at −70° C. To this wasadded triisopropyl borate (2.5 g, 13.30 mmol, 1.19 equiv) dropwise withstirring at −70° C. The mixture was warmed to 0° C., the reaction wasthen quenched by the addition of 13 mL of saturated ammonium chlorideand 3.4 mL of water. Phosphoric acid (85 wt %, 1.5 g, 1.2 equiv) wasadded and the mixture was stirred for 30 min. The organic layer wasseparated and dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was dissolved in 20 mL of toluene. The product wasprecipitated by the addition of 80 mL of heptane. The solids were washedwith 20 mL of heptane and collected by filtration. This resulted in 2.9g (85%) of 3-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenylboronic acidas a white solid.

Intermediate 60.3: 6-chloroquinazoline-2,4(1H,3H)-dione

Into a 500-mL 3-necked round-bottom flask, was placed a solution of2-amino-5-chlorobenzoic acid (10 g, 58.48 mmol, 1.00 equiv) in water(100 mL), acetic acid (8 g, 133.33 mmol, 2.24 equiv). This was followedby the addition of NaOCN (8.2 g, 126.15 mmol, 2.13 equiv). The mixturewas stirred for 30 mins at 30° C. To this was added sodium hydroxide (86g, 2.15 mol, 37.00 equiv). The resulting solution was stirred overnightat 30° C. The solids were collected by filtration. The residue wasdissolved in water. The pH value of the solution was adjusted to 7 withhydrogen chloride (12 mol/L). The solids were collected by filtration.This resulted in 5 g (44%) of 6-chloroquinazoline-2,4(1H,3H)-dione as awhite solid.

Intermediate 60.4: 2,4,6-trichloroquinazoline

Into a 50-mL round-bottom flask, was placed a solution of6-chloroquinazoline-2,4(1H,3H)-dione (2.2 g, 11.22 mmol, 1.00 equiv) in1,4-dioxane (20 mL), phosphoryl trichloride (17 g, 111.84 mmol, 10.00equiv). The resulting solution was stirred overnight at 120° C. in anoil bath. The resulting mixture was concentrated under vacuum. Thereaction was then quenched by the addition of 200 mL of water. Theresulting solution was extracted with 3×200 mL of ethyl acetate and theorganic layers combined. The residue was applied onto a silica gelcolumn with ethyl acetate/petroleum ether (1:50). This resulted in 1.8 g(69%) of 2,4,6-trichloroquinazoline as a white solid.

Intermediate 60.5: tert-butyl4-(3-(2,6-dichloroquinazolin-4-yl)phenyl)piperazine-1-carboxylate

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of3-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenylboronic acid(intermediate 60.2) (960 mg, 3.14 mmol, 1.00 equiv),2,4,6-trichloroquinazoline (800 mg, 3.43 mmol, 1.09 equiv),PdCl₂(dppf).CH₂Cl₂ (130 mg, 0.16 mmol, 0.05 equiv), Potassium Carbonate(860 mg, 6.23 mmol, 1.99 equiv) in N,N-dimethylformamide (30 mL). Theresulting solution was stirred for 3 h at 85° C. The reaction was thenquenched by the addition of 50 mL of saturated brine. The resultingsolution was extracted with 2×30 mL of ethyl acetate and the organiclayers combined and dried over anhydrous sodium sulfate and concentratedunder vacuum. The residue was applied onto a silica gel column withethyl acetate/petroleum ether (1:6). This resulted in 0.45 g (31%) oftert-butyl4-(3-(2,6-dichloroquinazolin-4-yl)phenyl)piperazine-1-carboxylate as ayellow solid.

Intermediate 60.6: 2,6-dichloro-4-(3-(piperazin-1-yl)phenyl)quinazoline2,2,2-trifluoroacetate

To intermediate 60.5 (100 mg, 0.22 mmol, 1.00 equiv) was addeddichloromethane (10 mL) and 2,2,2-trifluoroacetic acid (124 mg, 1.09mmol, 5.00 equiv) and the resulting solution was stirred for 3 h at 40°C. The reaction was then concentrated under vacuum to afford 70 mg ofIntermediate 60.6 as yellow solid.

Intermediate 60.7: tert-butyl(4(3-(2,6-dichloroquinazolin-4-yl)phenyl)piperazin-1-yl)methanediylidenedicarbamate

To Intermediate 60.6 (70 mg, 0.15 mmol, 1.00 equiv) in dichloromethane(10 mL) was addedN-tert-butoxycarbonyl-N′-tert-butoxycarbonyl-N″-trifluoromethanesulfonylguanidine(91 mg, 0.23 mmol, 1.57 equiv) and triethylamine (38 mg, 0.38 mmol, 2.54equiv) and the resulting solution was stirred for 3 h at 40° C. Themixture was then concentrated under vacuum, the residue applied onto asilica gel column and eluted with ethyl acetate/petroleum ether (1:8) toafford 70 mg (77%) of Intermediate 60.7 as a yellow solid.

Intermediate 60.8: tert-butyl(4-(3-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)methanediylidenedicarbamate

To Intermediate 60.7 (70 mg, 0.12 mmol, 1.00 equiv) in NMP (1.5 mL) wasadded guanidine (0.24 mL, 2.00 equiv, 1 mol/L) and1,4-diaza-bicyclo[2.2.2]octane (26 mg, 0.23 mmol, 1.99 equiv) and theresulting solution stirred for 1.5 h at 25° C. The reaction was quenchedby the addition of 20 mL of water and the resulting solution wasextracted with 2×20 mL of ethyl acetate. The organic layers werecombined, dried over anhydrous sodium sulfate, concentrated, the residueapplied onto a silica gel column and eluted withdichloromethane/methanol (5:1) to afford 30 mg (41%) of Intermediate60.8 as a yellow solid.

Compound 60:4-(3-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazine-1-carboximidamide

To Intermediate 60.8 (30 mg, 0.05 mmol, 1.00 equiv) in dichloromethane(5 mL) was added 2,2,2-trifluoroacetic acid (0.2 mL) and the resultingsolution stirred for 6 h at 30° C. The mixture was then concentratedunder vacuum and the residue lyophilized to afford 20 mg (75%) of a TFAsalt of the title compound as an off-white solid. ¹H-NMR (300 MHz,CD₃OD, ppm): 7.97-8.08 (m, 3H), 7.54-7.59 (m, 1H), 7.28-7.39 (m, 3H),3.71 (d, 0.1=−4.8 Hz, 4H), 3.44 (d, J=4.8 Hz, 4H). MS (ES, m/z): 424.0[M+H]⁺.

Example 612-(4-(4-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)aceticacid

Intermediate 61.1: 2,6-dichloro-4-(4-(piperazin-1-yl)phenyl)quinazolinehydrochloride

Following the procedures outlined in example 60, substituting1,4-dibromobenzene for 1,3-dibromobenzene,2,6-dichloro-4-(4-(piperazin-1-yl)phenyl)quinazoline hydrochloride wasobtained as a red solid.

Intermediate 61.2: methyl2-(4-(4-(2,6-dichloroquinazolin-4-yl)phenyl)piperazin-1-yl)acetate

To methyl 2-bromoacetate (116 mg, 0.76 mmol, 3.00 equiv) inN,N-dimethylformamide (10 mL) was added potassium carbonate (140 mg,1.01 mmol, 4.00 equiv) followed by the portion-wise addition ofIntermediate 61.1 (100 mg, 0.25 mmol, 1.00 equiv) and the reaction wasstirred for 4 h at 30° C. The mixture was concentrated under vacuum andthe residue applied onto a silica gel column, eluting with ethylacetate/petroleum ether (1:5) to afford 60 mg (55%) of Intermediate 61.2as a yellow solid.

Intermediate 61.3: methyl2-(4-(4-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)acetate

To Intermediate 61.2 (60 mg, 0.14 mmol, 1.00 equiv) in NMP (5 mL) wasadded 1,4-diaza-bicyclo[2.2.2]octane (DABCO; 15 mg, 0.13 mmol, 1.00equiv), guanidine (0.3 mL of a 1M solution in NMP, 2.00 equiv) and theresulting solution was stirred for 2 h at 30° C. The reaction wasdiluted with 10 mL of water, extracted with 4×10 mL of ethyl acetate andthe organic layers combined and dried over anhydrous sodium sulfate andthen concentrated under vacuum. The residue was applied onto a silicagel column and eluted with dichloromethane/methanol (50:1-20:1) toafford 30 mg (47%) of Intermediate 61.3 as a yellow solid.

Compound 61:2-(4-(4-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)aceticacid

To Intermediate 61.3 (20 mg, 0.04 mmol, 1.00 equiv) in methanol (5 mL)was added a solution of LiOH (32 mg, 1.33 mmol, 30.00 equiv) in water (1mL) and the reaction was stirred for 3 h at 25° C. The solution wasconcentrated under vacuum, the pH value adjusted to 6 with aqueous HCl(1 mol/L) and the resulting solids were collected by filtration toafford 15.6 mg (80%) of compound 61 as a yellow solid. ¹H-NMR (300 MHz,DMSO ppm): 8.07-8.06 (t, 1H), 7.96-7.93 (t, 2H), 7.72-7.69 (d, J=8.7 Hz,2H), 7.22-7.19 (d, J=8.7 Hz, 2H), 3.58-3.54 (m, 4H), 3.43-3.36 (m, 6H).MS (ES, m/z): 440 [M+H]⁺.

Example 622-(4-(3-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)aceticacid

Compound 62:2-(4-(3-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)aceticacid

Compound 62 was prepared from intermediate 60.6, using the proceduresdescribed for Example 61. ¹H-NMR (300 HHz, DMSO-d₆, ppm): 7.80-7.86 (m,3H), 7.41-7.46 (m, 1H), 7.16-7.22 (m, 2H), 7.08-7.10 (m, 1H), 3.13 (brs,4H), 2.71 (brs, 4H). MS (ES, m/z): 440 [M+H]⁺;

Example 632-(6-chloro-4-(3-(4-((2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)piperazin-1-yl)phenyl)quinazolin-2-yl)guanidine

Intermediate 63.1: (2R,3S,4R,5R)-2,3,4,5,6-pentaacetoxyhexanoic acid

Into a 50-mL 3-necked round-bottom flask, was placed ZnCl₂ (0.5 g, 0.50equiv), acetic anhydride (5 mL). To the above was added sodium(2S,3R,4S,5R)-2,3,4,5,6-pentahydroxyhexanoate (1.6 g, 6.97 mmol, 1.00equiv, 95%) at −5° C. Anhydrous HCl was introduced in for 0.5 h at 0° C.The resulting solution was stirred overnight at room temperature. Thereaction mixture was cooled to 0° C. The reaction was then quenched bythe addition of 8 g of ice. The mixture was stirred for 1 h at roomtemperature. The resulting solution was diluted with 20 mL of water. Theresulting solution was extracted with 3×20 mL of dichloromethane and theorganic layers combined and dried over anhydrous sodium sulfate andconcentrated under vacuum. This resulted in 1.0 g (35%) of(2R,3S,4R,5R)-2,3,4,5,6-pentaacetoxyhexanoic acid as a yellow liquid.

Intermediate 63.2: (2R,3R,4S,5R)-6-chloro-6-oxohexane-1,2,3,4,5-pentaylpentaacetate

Into a 50-mL 3-necked round-bottom flask, was placed a solution of(2R,3S,4R,5R)-2,3,4,5,6-pentaacetoxyhexanoic acid (intermediate 63.1)(610 mg, 1.35 mmol, 1.00 equiv, 90%) in CCl₄ (30 mL). This was followedby the addition of oxalyl dichloride (3 mL) dropwise with stirring. Theresulting solution was heated to reflux for 3 h in an oil bath. Theresulting mixture was concentrated under vacuum. This resulted in 0.62 g(crude) of intermediate 63.2 as yellow oil.

Intermediate 63.3:2-(6-chloro-4-(3-(4-((2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)piperazin-1-yl)phenyl)quinazolin-2-yl)guanidine2,2,2-trifluoroacetate

To Intermediate 60.6 (150 mg, 0.32 mmol, 1.00 equiv) in dichloromethane(5 mL) was added triethylamine (96 mg, 0.95 mmol, 2.99 equiv) and thesolution cooled to 0° C. Intermediate 63.2 (407 mg, 0.96 mmol, 3.02equiv) in dichloromethane (5 mL) was then added dropwise and thereaction was stirred for 1 h at room temperature. The resulting mixturewas concentrated under vacuum, the residue applied onto a silica gelcolumn and then eluted with ethyl acetate/petroleum ether (1:2) toafford 150 mg (62%) of Intermediate 63.3 as a yellow solid.

Intermediate 63.4:(2R,3R,4S,5R)-6-(4-(3-(6-chloro-2-(diaminomethyleneamino)-quinazolin-4-yl)phenyl)piperazin-1-yl)-6-oxohexane-1,2,3,4,5-pentaylpentaacetate

To Intermediate 63.3 (150 mg, 0.20 mmol, 1.00 equiv) in NMP (5 mL) wasadded guanidine (0.8 mL of a 1 mol/L solution in NMP; 4.0 equiv) and1,4-diaza-bicyclo[2.2.2]octane (DABCO: 44.8 mg, 0.40 mmol, 2.00 equiv)and the resulting solution was stirred for 1.5 h at 30° C. The reactionwas quenched by the addition of 10 mL of water and then extracted with2×10 mL of ethyl acetate. The organic layers combined, dried overanhydrous sodium sulfate, concentrated, applied onto a silica gel columnand then eluted with dichloromethane/methanol (10:1) to afford 30 mg(19%) of Intermediate 63.4 as a yellow solid.

Compound 63:2-(6-chloro-4-(3-(4-((2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)piperazin-1-yl)phenyl)quinazolin-2-yl)guanidine

To Intermediate 63.4 (25 mg, 0.03 mmol, 1.00 equiv) in methanol (5 mL),was added a solution of LiOH (3.9 mg, 0.16 mmol, 5.03 equiv) in water(0.2 mL) and the resulting solution was stirred for 0.5 h at 0° C. ThepH value of the solution was adjusted to 7 with aqueous HCl (5%), theresulting mixture was concentrated under vacuum and then purified byPrep-HPLC to afford 10 mg (45%) a TFA salt of compound 63 as a yellowsolid. LCMS (ES, m/z): 560.0 [M+H]⁺: ¹H-NMR (300 MHz, CD₃OD, ppm):7.96-8.09 (m, 3H), 7.52-7.57 (m, 1H), 7.25-7.39 (m, 3H), 4.73 (d, J=5.1Hz, 1H), 4.07-4.09 (m, 1H), 3.62-3.89 (m, 8H). MS (ES, m/z): 560.0[M+H]⁺

Example 643-(4-(3-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)propanoicacid

Intermediate 64.1: methyl3-(4-(3-(2,6-dichloroquinazolin-4-yl)phenyl)piperazin-1-yl)propanoate

To Intermediate 60.6 (200 mg, 0.51 mmol, 1.00 equiv) in tetrahydrofuran(10 mL) was added methyl acrylate (253 mg, 2.94 mmol, 5.81 equiv) andtriethylamine (253 mg, 2.50 mmol, 4.95 equiv) and the resulting mixturewas stirred for 3 h at room temperature. The reaction was concentratedunder vacuum, the residue applied onto a silica gel column and theneluted with ethyl acetate/petroleum ether (1:3) to afford 100 mg (44%)of Intermediate 64.1 as a yellow solid.

Compound 64:3-(4-(3-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)propanoicacid

Compound 64 was prepared from Intermediate 64.1 using the proceduresdescribed in Example 61, affording 25 mg of the title compound as ayellow solid. ¹H-NMR (300 MHz. DMSO-d₆, ppm): δ 7.89-7.92 (m, 3H),7.42-7.47 (m, 1H), 7.35 (brs, 1H), 7.15-7.24 (m, 2H), 3.25 (brs, 4H),2.63-2.74 (m, 6H), 2.31-2.35 (m, 2H). LCMS (ES, m/z): 454.0 [M+H]⁺

Example 651-(4-(3-(4-(3-aminopropyl)piperazin-1-yl)phenyl)-6-chloroquinazolin-2-yl)guanidine

Compound 65:1-(4-(3-(4-(3-aminopropyl)piperazin-1-yl)phenyl)-6-chloroquinazolin-2-yl)guanidine

A hydrochloride salt of the title compound was prepared using proceduressimilar to those outlined in Example 61, starting with intermediate 60.6and tert-butyl 3-bromopropylcarbamate. MS (ES, m/z): 439 [M+H]⁺

Example 664-(4-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazine-1-carboximidamide

Compound 66:4-(4-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazine-1-carboximidamide

A TFA salt of Compound 66 was prepared from Intermediate 61.1, using theprocedures described in Example 60. MS (ES, m/z): 424 [M+H]⁺

Example 672-(4-(3-(4-(3-guanidinopropyl)piperazin-1-yl)phenyl)-6-chloroquinazolin-2-yl)guanidine

Compound 67:2-(4-(3-(4-(3-guanidinopropyl)piperazin-1-yl)phenyl)-6-chloroquinazolin-2-yl)guanidine

A hydrochloride salt of Compound 67 was prepared from Compound 65 usingthe procedures outlined in Example 60. MS (ES, m/z): 481 [M+H]⁺

Example 682-(6-chloro-4-(3-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)quinazolin-2-yl)guanidine

Compound 68:2-(6-chloro-4-(3-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)quinazolin-2-yl)guanidine

A TFA salt of Compound 68 was prepared from Compound 60.6 and ethyleneoxide using the procedures outlined in Example 61. MS (ES, m/z): 426[M+H]⁺

Example 692-(6-chloro-4-(4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)quinazolin-2-yl)guanidine

Compound 69:2-(6-chloro-4-(4-(4-(2-hydroxyethyl)piperazin-1-yl)phenyl)quinazolin-2-yl)guanidine

a TFA salt of Compound 69 was prepared from Intermediate 61.1 using theprocedures described in Example 68. MS (ES, m/z): 426 [M+H]⁺

Example 704-(4-(3-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)butanoicacid 2,2,2-trifluoroacetic acid salt

Compound 70:4-(4-(3-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)butanoicacid

Compound 70 was prepared from Intermediate 60.6 and methyl4-bromobutanoate using the procedures described in Example 61.Purification by silica gel column with methanol:water (0˜0.04) gave aTFA salt of the title compound as a yellow solid. ¹H-NMR (300 MHz, DMSO,ppm): δ 11.33 (s, 1H), 8.09-8.19 (m, 2H), 7.96-7.96 (s, 1H), 7.53-7.58(m, 1H), 7.25-7.37 (m, 3H), 4.0 (s, 4H), 3.16 (s, 6H), 2.34-2.39 (m,2H), 1.92 (s, 2H); MS (ES, m/z): 468 [M+H]

Examples 71-104

Examples 71-104 were prepared using methods described in Examples 1-70.Characterization data (mass spectra) for compounds 71-104 are providedin Table 3.

Example 71(E)-3-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)propane-1-sulfonicacid

Example 722-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)-N-(phosphonomethyl)phenylsulfonamido)aceticacid

Example 734-(4-(4-(6-chloro-2-(diaminomethyleneamino)quinazolin-4-yl)phenyl)piperazin-1-yl)butanoicacid

Example 74 (E)-N-(diaminomethylene)-3(4(N-(ethylcarbamoyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylamide

Example 75 (E)-N-(diaminomethylene)-3-(4(4-(N-(2-(dimethylamino)ethylcarbamoyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylamide

Example 764-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)phenylphosphonicacid

Example 77(E)-N-(diaminomethylene-3,5-difluoro-4-(4-(N-methyl-N-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)sulfamoyl)phenoxy)phenyl)-2-methylacrylamide

Example 783-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)propane-1-sulfonicacid

Example 792-(4-(4-(4-(3-aminopropyl)piperazin-1-yl)pbenyl)-6-chloroquinazolin-2-yl)guanidine

Example 803-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)-N-(2-(2-(2-(2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)benzenesulfonamide

Example 81N,N′-(2,2′-(2,2-(2,2′-(2,2′-(4,4′-oxybis(methylene)bis(1H-1,2,3-triazole-4,1-diyl))bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Example 82N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Example 831-(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazole-4,5-dicarboxylicacid

Example 84(E)-3-(4-(4-(N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-N-(diaminomethylene)-2-methylacrylamide

Example 852-(4-(4-(4-(2-aminoethyl)piperazin-1-yl)phenyl)-6-chloroquinazolin-2-yl)guanidine

Example 86(E)-3-(4-(4-(N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethylcarbamoyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-N-(diaminomethylene)-2-methylacrylamide

Example 87N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,34-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Example 88N1,N4-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolino-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Example 89 1-(4-(4-(4(3-guanidinopropyl)piperazin-1-yl)phenyl)-6-chloroquinazolin-2-yl)guanidine

Example 90(E)-2-(4-(2-(4-(4-(3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)ethyl)piperazin-1-yl)aceticacid

Example 91N-(1-amino-1-imino-5,8,11-trioxa-2-azatridecan-13-yl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Example 92N-(1-amino-1-imino-5,8,11-trioxa-2-azatridecan-13-yl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Example 93(E)-1-(3-(3,5-difluoro-4-phenoxyphenyl)-2-methylallyl)guanidine

Intermediate 93.1(E)-3-(3,5-difluoro-4-phenoxyphenyl)-2-methylprop-2-en-1-ol

To a solution of (E)-ethyl3-(3,5-difluoro-4-phenoxyphenyl)-2-methylacrylate (Intermediate 41.2)(800 mg, 2.51 mmol) in dry DCM (25 mL) under N2 at −78° C. was added asolution of DIBAL-H (8.79 mL, 1M in DCM) dropwise over several minutes.The reaction was allowed to warm to room temperature over 2 hours. Thereaction mixture was cooled to 0° C., quenched with 25 mL of Rochelle'sSalt solution (10% w/v in water), and stirred vigorously for 1 hour. Theresulting suspension was diluted with water (20 mL) and extracted withDCM (3×30 mL). The combined organic layers were dried over Na₂SO₄ andconcentrated. The resulting oil was applied onto a silica gel column(50% EtOAc in hexanes) to yield 566 mg of the title compound (82%) as ayellow oil.

Intermediate 932(E)-2-(3-(3,5-difluoro-4-phenoxyphenyl)-2-methylallyl)isoindoline-1,3-dione

To a solution of(E)-3-(3,5-difluoro-4-phenoxyphenyl)-2-methylprop-2-en-1-ol(Intermediate 93.1) (410 mg, 1.49 mmol) in dry toluene (7.45 mL) underN2 was added PPh₃ and phthalimide. The resulting solution was cooled to0° C. and diethyl azodicarboxylate (DEAD, 0.748 mL) was added dropwiseover several minutes. The reaction was allowed to warm to roomtemperature and stirred overnight. After diluting with EtOAc (20 mL),the organic layer was washed with water (2×30 mL), brine (30 mL) anddried over Na₂SO₄. After removal of solvent, the resulting residue wasapplied to a silica gel column (15% EtOAc in hexanes) to yield 385 mg ofthe title compound (63%) as an oil.

Intermediate 93.3(E)-3-(3,5-difluoro-4-phenoxyphenyl)-2-methylprop-2-en-1-amine

To a solution of(E)-2-(3-(3,5-difluoro-4-phenoxyphenyl)-2-methylallyl)isoindoline-1,3-dione(Intermediate 93.2, 100 mg, 0.25 mmol) in methanol (1 mL) was addedhydrazine hydrate (25 mg, 0.5 mmol) and the reaction stirred at 50° C.overnight. The white solid was filtered with DCM, and the solventremoved from the filtrate. The residue was brought up in DCM andfiltered. This was repeated until no further precipitate formed to give49.5 mg of the title compound (71%) as a yellow oil, a 10 mg portion ofwhich was diluted with 1N HCl and freeze dried to give 7.8 mg of thetitle compound as an HCl salt. ¹H-NMR (400 MHz, d₆-DMSO): δ 8.25 (s,2H), 7.37 (t, 2H), 7.20 (d, 2H), 7.12 (t, 1H), 6.97 (s, 1H), 3.57 (s,2H), 1.96 (s, 3H). MS (m/z): 258.96 (M-NH₂).

Intermediate 93.4:(E)-4-(4-(3-amino-2-methylprop-1-enyl)-2,6-difluorophenoxy)-N-(2-(dimethylamino)ethyl)benzenesulfonamide

To a solution of(E)-3-(3,5-difluoro-4-phenoxyphenyl)-2-methylprop-2-en-1-amine(intermediate 93.3, 100 mg, 0.364 mmol) in DCM (0.364 mL, 1M) was addedchlorosulfonic acid (2.91 mmol, 194.3 uL) in 4 portions dropwise every20 minutes. The reaction was stirred an additional 20 minutes and thenquenched into a solution of N1,N1-dimethylethane-1,2-diamine (3.78 mL)in DCM (12 mL) at 0° C. The resulting solution was warmed to roomtemperature and stirred for 30 minutes. Upon completion the solvent wasremoved and the residue brought up in 1:1 Acetonitrile:water solutionand purified by preparative HPLC to give 74.5 mg of the title compound(31%) as a TFA salt.

Compound 93:(E)-4-(2,6-difluoro-4-(3-guanidino-2-methylprop-1-enyl)phenoxy)-N-(2-(dimethylamino)ethyl)benzenesulfonamide

To a solution of(E)-4-(4-(3-amino-2-methylprop-1-enyl)-2,6-difluorophenoxy)-N-(2-(dimethylamino)ethyl)benzenesulfonamide(Intermediate 93.4, 39.3 mg, 0.092 mmol) in dry THF (460 uL, 0.2M) underN2 was added TEA (0.276 mmol, 27.9 mg) and(1H-pyrazol-1-yl)methanediamine hydrochloride (0.102 mmol, 14.9 mg). Theresulting solution was stirred for 1 hour, at which point LCMS indicatedcomplete conversion. The solvent was removed and the resulting residuebrought up in 1:1 ACN:water and purified by preparative HPLC to give16.9 mg of the title compound (26%) as a TFA salt. ¹H-NMR (400 MHz,CD₄OD): δ 7.87 (d, 2H), 7.12 (d, 2H), 7.08 (d, 2H), 3.92 (s, 2H), 3.62(m, 2H), 3.29 (m, 2H), 3.17 (t, 2H), 2.01 (s, 6H), 1.91 (s, 3H). MS(m/z): 468.12 (M+H)⁺.

Example 94N-(2-(2-(2-(2-(4,5-bis(hydroxymethyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Example 95N-(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)acetamide

Example 96N-(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)acetamide

Example 97N1,N31-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-4,7,10,13,16,19,22,25,28-nonaoxahentriacontane-1,31-diamide

Example 98N1,N31-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-4,7,10,13,16,19,22,25,28-nonaoxahentriacontane-1,31-diamide

Example 99(E)-3-(4-(4-(N-(1-amino-1-imino-5,8,11-trioxa-2-azatridecan-13-yl)sulfamoyl)phenoxy)-3,5-difuorophenyl)-N-(diaminomethylene)-2-methylacrylamide34

Example 100N,N′-(13-oxo-3,6,9,17,20,23-hexaoxa-12,14-diazapentacosane-1,25-diyl)bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Example 101(E)-N-(diaminomethylene)-3-(3,5-difluoro-4-(4-(N-(2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)sulfamoyl)phenoxy)phenyl)-2-methylacrylamide

Example 102 N1,N31-bis(2-(2-(2-(2-(4-(4-((E)-3(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-4,7,10,13,16,19,22,25,28-nonaoxahentriacontane-1,31-diamide

Example 103N,N′-(13-oxo-3,6,9,17,20,23-hexaoxa-12,14-diazapentacosane-1,25-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Example 104N1,N4-bis(20-(4-(4-((E)-3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)-3,6,9,12,15,18-hexaoxaicosyl)-2,3-dihydroxysuccinamide

TABLE 3 Analytical Data for Example Compounds 71-104 Example [M + H]⁺ 71533 72 523 73 468 74 482 75 525 76 527 77 589 78 493 79 439 80 628 811239.1 82 546.3 83 686 84 542 85 425 86 629 87 604 [M + 2]/2 88 604 [M +2]/2 89 481 90 581 91 588 92 588 94 658 95 588 96 588 97 1571 98 1571 99628 100  1117 101  628 102  1649 103  1117 104  1549

Example 1054-/3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)-polyethylimino-sulfonamide

Example 105 is prepared from polyethylamine according to the proceduresin described in Examples 1-70, where “x,” “y,” “n” and “m” aredetermined by the stoichiometry of the sulfonylchloride andpolyethylamine.

Example 106

As illustrated below, other polymeric nucleophiles are employed usingthe procedures described in Examples 1-70 to prepare polyvalentcompounds:

Example 107

As illustrated below, polymeric electrophiles are used with nucleophilicIntermediates to prepare polyvalent compounds using, for example, theprocedures outlined in Example 68.

Example 108-147

General Procedure for copolymerization of Intermediate 108.1 andIntermediate 108.2 with other monomers

Intermediate 108.1:N-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)acrylamide

Intermediate 108.1 (Int 108.1) was prepared from intermediate 30.7 andacrylic acid using procedures described in Examples 1-70. MS (m/z):361.1 (M+H)

Intermediate 108.2:N-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)ethyl)acrylamide

Intermediate 108.2 (Int 108.2) was prepared from intermediate 30.7 usingprocedures described in Examples 1-70. MS (m/z): 404.1 (M+H)

A 20-mL vial is charged with a total of 1 g of Intermediate 108.1 orIntermediate 108.2 and other monomers, a total of 9 g ofisopropanol/dimethylformamide solvent mixture, and 20 mg ofazobisisobutyronitrile. The mixture is degassed for 1 min and is sealedunder a nitrogen atmosphere. The stoichiometry for each example is shownin Table 1. The reaction mixture is heated in an oil bath to 70° C.under stirring. After 8 h at 70° C. the reaction mixture is cooled downto ambient temperature and then 10 mL of water is added. The solution isthen transferred to a dialysis bag (MWCO 1000) for dialysis against DIwater for 2 days. The resulting solution is freeze-dried to affordcopolymers.

TABLE 4 Examples of conditions that can be used to create copolymersfrom acrylamide-functionalized NHE inhibitors and substitutedacrylamides and methacrylates Monomer (mg) Int Poly(ethylene 108.1glycol) Ex- Or methyl am- Int acyl ether butyl acrylic Solvent (g) ple108.2 amide methacrylate acrylate acid IPA/DMF 108 10 990 0 0 0 0/9 10950 950 0 0 0 0/9 110 100 900 0 0 0 0/9 111 750 750 0 0 0 0/9 112 500 5000 0 0 0/9 113 10 990 0 0 0 2.25/6.75 114 50 950 0 0 0 2.25/6.75 115 100900 0 0 0 2.25/6.75 116 250 750 0 0 0 2.25/6.75 117 500 500 0 0 02.25/6.75 118 10 990 0 0 0  4.5/4.5 119 50 950 0 0 0  4.5/4.5 120 100900 0 0 0  4.5/4.5 121 250 750 0 0 0  4.5/4.5 122 500 500 0 0 0  4.5/4.5123 10 990 0 0 0 6.75/2.25 124 50 950 0 0 0 6.75/2.25 125 100 900 0 0 06.75/2.25 126 250 750 0 0 0 6.75/2.25 127 500 500 0 0 0 6.75/2.25 128 10990 0 0 0   9/0 129 50 950 0 0 0   9/0 130 100 900 0 0 0   9/0 131 250750 0 0 0   9/0 132 500 500 0 0 0   9/0 133 10 0 990 0 0 6.75/2.25 13450 0 950 0 0 6.75/2.25 135 100 0 900 0 0 6.75/2.25 136 250 0 750 0 06.75/2.25 137 500 0 500 0 0 6.75/2.25 138 100 775 0 25 0 6.75/2.25 139100 750 0 50 0 6.75/2.25 140 100 700 0 100 0 6.75/2.25 141 100 650 0 1500 6.75/2.25 142 100 600 0 200 0 6.75/2.25 143 100 800 0 0 10 6.75/2.25144 100 800 0 0 25 6.75/2.25 145 100 800 0 0 50 6.75/2.25 146 100 800 00 100 6.75/2.25 147 100 800 0 0 150 6.75/2.25

Example 148 Synthesis of 2-Methyl-acrylic acid3-trimethylsilanyl-prop-2-ynyl ester

A solution of trimethylsilyl propyn-1-ol (1 g, 7.8 mmol) and Et₃N (1.4mL, 10 mmol) in Et₂O (10 mL) is cooled to −20° C. and a solution ofmethacryloyl chloride (0.9 mL, 9.3 mmol) in Et₂O (5 mL) is addeddropwise over 1 h. The mixture is stirred at this temperature for 30min, and then allowed to warm to ambient temperature overnight. Anyprecipitated ammonium salts can be removed by filtration, and volatilecomponents can be removed under reduced pressure. The crude product isthen purified by flash chromatography.

Examples 149-154 General Procedure for synthesis of polyN-(2-hydroxypropyl)methacrylamide-co-prop-2-ynyl methacrylate

General procedure for copolymerization ofN-(2-hydroxypropyl)methacrylamide and 3-(trimethylsilyl)prop-2-ynylmethacrylate

A 100-mL round bottom flask equipped with a reflux condenser is chargedwith a total 5 g of N-(2-hydroxypropyl)methacrylamide and3-(trimethylsilyl)prop-2-ynyl methacrylate, 45 g ofisopropanol/dimethylformamide solvent mixture, and 100 mg ofazobisisobutyronitrile. The mixture is degassed for 1 min and maintainedunder nitrogen atmosphere during the reaction. Stoichiometry for eachexample is shown in Table 5. The reaction mixture is heated in an oilbath to 70° C. under stirring, and after 8 h the reaction mixture iscooled to ambient temperature and then 30 mL of solvent is evaporatedunder vacuum. The resulting solution is then precipitated into 250 mL ofEt₂O. The precipitate is collected, redissolved in 10 mL of DMF, andprecipitated again into 250 mL of Et₂. The resulting precipitate isdried under vacuum to afford copolymers.

General Procedure for Removal of Trimethyl Silyl Group

The trimethyl silyl protected polymer (4 g), acetic acid (1.5 equiv.mol/mol with respect to the alkyne-trimethylsilyl groups), and 200 mL ofTHF is mixed in a 500 mL flask. The mixture is cooled to −20° C. undernitrogen atmosphere and followed by addition of 0.20 M solution oftetra-n-butylammonium fluoride trihydrate (TBAF.3H₂O) in THF (1.5 equiv.mol/mol with respect to the alkyne-trimethylsilyl groups) over a courseof 5 min. The solution is stirred at this temperature for 30 min andthen warmed to ambient temperature for an additional 8 hours. Theresulting mixture is passed through a short silica pad and thenprecipitated in Et₂O. The resulting precipitate is dried under vacuum toafford copolymers.

TABLE 5 Examples of copolymerization conditions that can be used toprepared polymethacrylates Monomer (g) 3-(trimethylsilyl)N-(2-hydroxypropyl) prop-2-ynyl Solvent (g) Example methacrylamidemethacrylate IPA/DMF 149 2.5 2.5  0/45 150 2.5 2.5 11.25/33.75 151 2.52.5 22.5/22.5 152 2.5 2.5 33.75/11.25 153 2.5 2.5 45/0 

Examples 154-167 General Procedure for Post-Modification of Examples149-153 by [2+3] cycloaddition

Polymer 154 (54 mg) containing 0.1 mmol of alkyne moiety, a total of 0.1mmol of azido-compounds (Intermediate 28.1,13-azido-2,5,8,11-tetraoxatridecane,N-(2-azidoethyl)-3-(dimethylamino)propanamide and 1-azidodecane,corresponding ratios shown in Table 6), 0.05 mmol ofdiisopropylethylamine, and 1 mL of DMF is mixed at ambient temperatureand degassed for 1 min. While maintaining a nitrogen atmosphere, copperiodide (10 mg, 0.01 mmol) is then added to the mixture. The solution isstirred at ambient temperature for 3 days and then passed through ashort neutral alumina pad. The resulting solution is diluted with 10 mLof DI water, dialyzed against DI water for 2 days, and lyophilized toafford copolymers.

TABLE 6 Examples of compounds that can be prepared from polymericalkynes and varying ratios of substituted azides via [3 + 2]cycloaddition Azido compounds (mmol) Inter- 13-azido- N-(2-azidoethyl)-Exam- mediate 2,5,8,11- 3-(dimethylamino) 1- ple 28.1 tetraoxatridecanepropanamide azidodecane 155  0.002  0.098 0 0 156  0.005  0.095 0 0 1570.01 0.09 0 0 158  0.025  0.075 0 0 159 0.05 0.05 0 0 160 0.01  0.0880.002 0 161 0.01  0.085 0.005 0 162 0.01 0.08 0.01 0 163 0.01 0.07 0.020 164 0.01  0.088 0 0.002 165 0.01  0.085 0 0.005 166 0.01 0.08 0 0.01167 0.01 0.07 0 0.02

Example 168N1,N4-bis(2-(2-(23-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 168.1, bis(25-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate

To a 500 ml 3-necked roundbottom flask was added 2,3-dihydroxysuccinicacid (10.0 g, 66.62 mmol, 1.00 equiv), N,N′-Dicyclohexyl carbodiimide(DCC; 30.0 g, 145.42 mmol, 2.18 equiv) and tetrahydrofuran (THF; 100mL). This was followed by the addition of a solution ofN-hydroxysuccinimide (NHS; 16.5 g, 143.35 mmol, 2.15 equiv) in THF (100mL) at 0-10° C. The resulting solution was warmed to room temperatureand stirred for 16 h. The solids were filtered out and the filtrate wasconcentrated under vacuum. The crude product was re-crystallized fromN,N-dimethylformamide (DMF)/ethanol in the ratio of 1:10. This resultedin 5.2 g (22%) of the title compound as a white solid. ¹H-NMR (300 MHz,DMSO, ppm) δ 6.70 (d, J=7.8 Hz, 2H), 4.89 (d, J=−7.2 Hz, 2H), 2.89 (s,8H). MS (m/z): 367 [M+Na]⁺.

Intermediate 168.2N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

To a 50-mL 3-necked round-bottom flask was added2-(2-(2-aminoethoxy)ethoxy)ethanamine (3.2 g, 21.59 mmol, 21.09 equiv)and dichloromethane (DCM: 20 mL). This was followed by the addition of asolution of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (Intermediate 1.6) (400 mg, 1.02 mmol, 1.00 equiv) in DMF (5mL) dropwise with stirring. The resulting solution was stirred for 5 hat which time it was diluted with 100 mL of ethyl acetate. The resultingmixture was washed successively with 2×10 mL of water and 1×10 mL ofBrine. The organic layer was dried over anhydrous sodium sulfate andconcentrated under vacuum. This resulted in 300 mg (58%) ofN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas a yellow oil.

Compound 168,N1,N4-bis(2-(2-(2(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Into a 50-mL round-bottom flask was placed a solution ofN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(300 mg, 0.60 mmol, 1.00 equiv) in DMF (5 mL),bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (92.5 mg, 0.27mmol, 0.45 equiv) and triethylamine (TEA; 1.0 g, 9.88 mmol, 16.55equiv). The resulting solution was stirred overnight at room temperatureand then concentrated under vacuum. The crude product (300 mg) waspurified by Prep-HPLC with the following conditions: Column, SunFirePrep C18, 5 um, 19*150 mm; mobile phase. Water with 0.05% TFA and CH₃CN(20% CH₃CN up to 40% in 5 min, up to 100% in 2 min): Detector, uv220&254 nm. This resulted in 192.4 mg (28%) of a TFA salt of the titlecompound as a white solid. ¹H-NMR (300 MHz, DMSO, ppm) δ 7.92 (d, J=7.8Hz, 2H), 7.82 (m, 2H), 7.67 (t, J=7.8 Hz, 2H), 7.57 (m, 2H), 7.55 (d,J=6.9 Hz, 2H), 6.86 (m, 2H), 4.84 (s, 2H), 4.79 (s, 2H), 4.54 (d, 2H),4.48 (s, 2H), 3.92 (m, 2H), 3.53 (m, 22H), 3.18 (s, 6H), 3.07 (t, J=5.4Hz, 4H). MS (m/z): 1119 [M+H]⁺.

Example 169N1,N4-bis(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethyl)-2,3-dihydroxysuccinamide

Intermediate 169.1,N-(2-aminoethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Into a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 1.6) (100 mg, 0.26 mmol, 1.00 equiv) in DCM (5mL). This was followed by the addition of a solution ofethane-1,2-diamine (307 mg, 5.11 mmol, 19.96 equiv) in DCM/DMF (10/1mL). The resulting solution was stirred for 5 h at room temperature. Themixture was concentrated under vacuum. The resulting solution wasdiluted with 50 mL of ethyl acetate and washed with 2×10 mL of water andthen 1×10 mL of Brine. The organic layer was dried over anhydrous sodiumsulfate and concentrated under vacuum to afford 90 mg (76%) ofN-(2-aminoethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas yellow oil.

Compound 169,N1,N4-bis(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroiso-quinolin-4-yl)phenylsulfonamido)ethyl)-2,3-dihydroxysuccinamide

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution ofN-(2-aminoethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(250 mg, 0.60 mmol, 1.00 equiv) in DMF (5 mL),bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxsuccinate (Intermediate 168.1)(92 mg, 0.27 mmol, 0.44 equiv) and triethylamine (280 mg, 2.77 mmol,4.55 equiv) and the resulting solution was stirred overnight at roomtemperature. The resulting mixture was concentrated under vacuum, theresidue diluted with 100 mL of ethyl acetate and then washed with 2×10mL of water. The organic layer was dried over anhydrous sodium sulfateand concentrated under vacuum. The crude product was purified byPrep-HPLC with the following conditions: Column, SunFire Prep C18, 5 um,19*150 mm; mobile phase, Water with 0.05% TFA and CH₃CN (25% CH₃CN up to35% in 5 min, up to 100% in 2.5 min); Detector, uv 220&254 nm. Thisresulted in 88.4 mg (15%) of a TFA salt ofN1,N4-bis(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethyl)-2,3-dihydroxysuccinamideas a white solid. ¹H-NMR (400 MHz, CD₃OD, ppm) δ 7.67 (d, J=7.6 Hz, 2H),7.61 (s, 2H), 7.44 (t, J=7.6 Hz, 2H), 7.37 (d, J=7.6 Hz, 2H), 7.25 (d,J=2 Hz, 2H), 6.72 (s, 2H), 4.33 (t, J==6.4 Hz, 2H), 4.30 (s, 2H), 3.64(m, 4H), 3.21 (s, 4H), 2.98 (m, 2H), 2.90 (m, 4H), 2.65 (m, 2H), 2.42(s, 6H). MS (m/z): 943 [M+H]⁺.

Example 170N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 170.1,3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride

Using procedures outlined in Example 1 to prepare intermediate 1.6,substituting N-(2,4-dichlorobenzyl)ethanamine for1-(2,4-dichlorophenyl)-N-methylmethanamine, the title compound wasprepared as a hydrochloride salt.

Intermediate 170.2N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

To 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (300 mg, 1.51 mmol,1.00 equiv) in DCM (10 mL) was added TEA (375 mg, 3.00 equiv) followedby the portionwise addition of3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (500 mg, 1.23 mmol, 1.00 equiv). The resulting solution wasstirred for 1 h at room temperature and then concentrated under vacuum.The residue was applied onto a silica gel column and eluted with ethylacetate/petroleum ether (1:2) to afford 0.4 g (41%) ofN-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas yellow oil.

Intermediate 170.3,N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Into a 100-mL round-bottom flask, was placedN-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(400 mg, 0.68 mmol, 1.00 equiv), triphenylphosphine (400 mg, 2.20equiv), THF (10 mL) and water (1 mL) and the reaction was stirredovernight at room temperature. The resulting mixture was concentratedunder vacuum and applied onto a preparative thin-layer chromatography(TLC) plate, eluting with DCM:methanol(5:1). This resulted in 350 mg(73%) ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas yellow oil.

Compound 170,N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Into a 50-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(100 mg, 0.18 mmol, 1.00 equiv) in DMF (3 mL),bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (Intermediate168.1) (25 mg, 0.07 mmol, 0.45 equiv) and triethylamine (75 mg, 4.50equiv). The resulting solution was stirred overnight at roomtemperature. The reaction progress was monitored by LCMS. The resultingmixture was concentrated under vacuum. The crude product was purified byFlash-Prep-HPLC with water: methanol (1:10-1:100). This resulted in 12.1mg (5%) of N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-ethyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamideas yellow oil. ¹H-NMR (300 MHz, DMSO, ppm): δ 7.70-7.60 (m, 8H),7.53-7.49 (m, 6H), 6.88 (s, 2H), 5.61-5.59 (m, 2H), 4.38 (m, 2H),4.24-4.22 (m, 2H), 3.78-3.72 (m, 2H), 3.58-3.48 (m, 2H), 3.43 (m, 7H),3.43-3.40 (m, 11H), 3.27-3.20 (m, 5H), 2.91-2.87 (m, 6H), 2.76-2.70 (m,2H), 2.61-2.55 (m, 3H), 1.04-0.99 (m, 6H). MS (m/z): 1235 [M+H]⁺.

Example 1713,3′-(2,2′-(2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(6,8-dichloro-1,2,3,4-tetrahydroisoquinoline-4,2-diyl))dianiline

Intermediate 171.1,2-(2,4-dichlorobenzylamino)-1-(3-nitrophenyl)ethanone

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of2-bromo-1-(3-nitrophenyl)ethanone (10.0 g, 41.15 mmol, 1.00 equiv) inTHF (150 mL), (2,4-dichlorophenyl)methanamine (7.16 g, 40.91 mmol, 1.00equiv) and triethylamine (5.96 g, 59.01 mmol, 1.50 equiv). The resultingsolution was stirred for 2 h at 25° C. The solids were filtered out. Thefiltrate was concentrated to dryness and used for next step, assumingtheoretical yield.

Intermediate 171.2, 2-(2,4-dichlorobenzylamino)-1-(3-nitrophenyl)ethanol

Into a 500-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of intermediate171.1 (40.91 mmol, 1.00 equiv) in methanol (150 mL). This was followedby the addition of NaBH₄ (2.5 g, 65.79 mmol, 1.50 equiv) in severalbatches at 0° C. The resulting solution was stirred for 2 h at 25° C.The reaction was then quenched by the addition of aqueous NH₄Cl. Theresulting mixture was concentrated under vacuum, and the solids werecollected by filtration. The crude product was purified byre-crystallization from ethyl acetate. This resulted in 3.5 g (23%) of2-(2,4-dichlorobenzylamino)-1-(3-nitrophenyl)ethanol as a yellowishsolid.

Intermediate 171.3,6,8-dichloro-4-(3-nitrophenyl)-1,2,3,4-tetrahydroisoquinoline

To 2-(2,4-dichlorobenzylamino)-1-(3-nitrophenyl)ethanol (intermediate171.2) (500 mg, 1.47 mmol, 1.00 equiv) in DCM (10 mL) was added conc.sulfuric acid (4 mL) dropwise with stirring at 0-5° C. The resultingsolution was stirred for 12 h at room temperature. The reaction was thenquenched by the addition of water/ice. The pH value of the solution wasadjusted to 10 with sodium hydroxide. The resulting solution wasextracted with 2×50 mL of DCM and the organic layers combined and driedover anhydrous sodium sulfate and concentrated under vacuum. Thisresulted in 300 mg (63%) of6,8-dichloro-4-(3-nitrophenyl)-1,2,3,4-tetrahydroisoquinoline as yellowoil.

Intermediate 171.4,2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate)

Into a 250-mL 3-necked round-bottom flask, was placed a solution oftetraethylene glycol (10 g, 51.55 mmol, 1.00 equiv) in DCM (100 mL).This was followed by the addition of a solution of4-methylbenzene-1-sulfonyl chloride (21.4 g, 112.63 mmol, 2.20 equiv) inDCM (50 mL) dropwise with stirring at 5° C. To this was addedN,N-dimethylpyridin-4-amine (15.7 g, 128.69 mmol, 2.50 equiv). Theresulting solution was stirred for 2 h at room temperature at which timeit was diluted with 100 mL of water. The resulting solution wasextracted with 3×100 mL of DCM and the organic layers combined. Theresulting mixture was washed with 1×100 mL of brine and thenconcentrated under vacuum. The residue was applied onto a silica gelcolumn and eluted with ethyl acetate/petroleum ether (1:2) to afford 11g (43%) of the title compound as white oil.

Intermediate 171.5,2,2′-(2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(6,8-dichloro-4-(3-nitrophenyl)-1,2,3,4-tetrahydroisoquinoline

To 6,8-dichloro-4-(3-nitrophenyl)-1,2,3,4-tetrahydroisoquinoline(intermediate 171.3) (171 mg, 0.53 mmol, 2.50 equiv) in DMF (2 mL) wasadded potassium carbonate (87 mg, 0.63 mmol, 3.00 equiv) andintermediate 171.4 (106 mg, 0.21 mmol, 1.00 equiv) and the resultingsolution was stirred at 50° C. After stirring overnight, the resultingsolution was diluted with 20 ml of water. The resulting mixture wasextracted with 3×20 ml of ethyl acetate and the organic layers combinedand concentrated under vacuum. The crude product was purified byPrep-HPLC with methanol:water (1:1). This resulted in 10 mg (2%) of2,2′-(2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(6,8-dichloro-4-(3-nitrophenyl)-1,2,3,4-tetrahydroisoquinoline)as a light yellow solid.

Compound 171,3,3′-(2,2,2′2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethane-2,1-diyl))bis(6,8-dichloro-1,2,3,4-tetrahydroisoquinoline-4,2-diyl))dianiline

To intermediate 171.5 (50 mg, 0.06 mmol, 1.00 equiv) in ethanol (5 mL)was added iron (34 mg, 0.61 mmol, 9.76 equiv) followed by the additionof hydrogen chloride (5 mL) dropwise with stirring. The resultingsolution was stirred for 2 h at room temperature and then for anadditional 4 h at 55° C. The reaction progress was monitored by LCMS.The solids were filtered out and the resulting solution was diluted with10 mL of water. The resulting mixture was concentrated under vacuum andthe pH of the solution was adjusted to 9-10 with sodium carbonate. Theresulting solution was extracted with 3×50 mL of ethyl acetate and theorganic layers combined, washed with 50 mL of brine and thenconcentrated under vacuum. The crude product was purified by Prep-HPLCwith H₂O:CH₃CN (10:1). This resulted in 5 mg (11%) of3,3′-(2,2′-(2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(ethane-2I-diyl))bis(6,8-dichloro-1,2,3,4-tetrahydroisoquinoline-4,2-diyl))dianilineas a yellow solid.). ¹H-NMR (400 MHz, CD₃OD, ppm) δ 7.27 (m, 2H), 7.06(m, 2H), 6.80 (s, 2H), 6.63 (d, 2H), 6.54 (m, 4H), 4.14 (m, 2H), 4.02(d, 2H), 3.65 (m, 12H), 3.19 (m, 3H), 2.81 (s, 4H), 2.71 (m, 2H). MS(m/z): 745 [M+H]⁺.

Example 172N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 28.1:N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

To 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (1.5 g, 6.87 mmol,1.79 equiv) in DCM (20 mL) was added triethylamine (1.5 g, 14.82 mmol,3.86 equiv) and3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (1.5 g, 3.84 mmol, 1.00 equiv). The reaction was stirredovernight at room temperature at which time the resulting mixture wasconcentrated under vacuum. The residue was dissolved in 100 mL of ethylacetate and then was washed with 2×20 mL of water, dried over anhydroussodium sulfate and concentrated under vacuum. This resulted in 1.8 g(85%) ofN-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas yellow oil.

Compound 28,N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

ToN-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(1.8 g, 3.26 mmol, 1.00) equiv) in THF (30 mL) was addedtriphenylphosphine (2.6 g, 9.91 mmol, 3.04 equiv). The resultingsolution was stirred overnight at room temperature and then concentratedunder vacuum. The crude product (5.0 g) was purified by Flash-Prep-HPLCwith the following conditions: Column, silica gel, mobile phase,methanol:water=1:9 increasing to methanol:water=9:1 within 30 min;Detector, UV 254 nm, 1.2 g product was obtained. This resulted in 1.2 g(64%) ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas yellow oil.

Compound 172,N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

ToN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(compound 28) (1.2 g, 2.28 mmol, 1.00 equiv) in DMF (8 mL) was addedbis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (intermediate168.1) (393 mg, 1.14 mmol, 0.50 equiv) and triethylamine (1.5 g, 14.82mmol, 6.50 equiv) and the resulting solution was stirred overnight atroom temperature. The mixture was concentrated under vacuum and thecrude product was purified by Flash-Prep-HPLC with the followingconditions: Column, silica gel; mobile phase, methanol:water=1:9increasing to methanol:water=9:1 within 30 min: Detector, UV 254 nm.This resulted in 591 mg (43%) ofN1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamideas a light yellow solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.92 (d, J=7.8Hz, 2H), 7.81 (m, 2H), 7.67 (t, J=7.8 Hz, 2H, 7.57 (m, 2H), 7.55 (d,J=6.9 Hz, 2H), 6.85 (m, 2H), 4.78 (s, 2H), 4.77 (s, 2H), 4.54 (d, J=40.2Hz, 2H), 4.48 (s, 2H), 3.92 (m, 2H), 3.53 (m, 30H), 3.18 (s, 6H), 3.07(t, J=5.4 Hz, 4H). MS (m/z): 603 [½M+H]⁺.

Example 173N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 173.1,N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline

Into a 10-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of4-(3-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline(intermediate 1.4) (400 mg, 1.08 mmol, 1.00 equiv) in DMSO (6 mL),2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (236.11 mg, 1.08 mmol,1.00 equiv), (S)-pyrrolidine-2-carboxylic acid (24.79 mg, 0.21 mmol,0.20 equiv), copper(I) iodide (20.48 mg, 0.11 mmol, 0.10 equiv) andpotassium carbonate (223.18 mg, 1.62 mmol, 1.50 equiv). The resultingsolution was stirred at 90° C. in an oil bath and the reaction progresswas monitored by LCMS. After stirring overnight the reaction mixture wascooled with a water/ice bath and then diluted with ice water. Theresulting solution was extracted with 3×30 mL of ethyl acetate and theorganic extracts were combined and washed with 2×20 mL of brine. Themixture was dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was applied onto a silica gel column with ethylacetate/petroleum ether (2:1). This resulted in 130 mg (24%) ofN-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenamineas yellow oil.

Intermediate 173.2,N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)aniline

Into a 50-mL round-bottom flask, was placed a solution of intermediate173.1 (350 mg, 0.69 mmol, 1.00 equiv) in THF/water (4/0.4 mL) andtriphenylphosphine (205 mg, 0.78 mmol, 1.20 equiv). The resultingsolution was stirred overnight at 40° C. in an oil bath. The resultingmixture was then concentrated under vacuum. The pH of the solution wasadjusted to 2-3 with 1N hydrogen chloride (10 ml). The resultingsolution was extracted with 2×10 mL of ethyl acetate and the aqueouslayers combined. The pH value of the solution was adjusted to 11 withNH₃.H₂O. The resulting solution was extracted with 3×30 mL of DCM andthe organic layers combined. The resulting mixture was washed with 30 mLof brine. The mixture was dried over anhydrous sodium sulfate andconcentrated under vacuum. This resulted in 250 mg (75%) ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)anilineas yellow oil.

Compound 173,N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

To intermediate 173.2 (240 mg, 0.50 mmol, 1.00 equiv) in DMF (5 mL) wasadded TEA (233 mg, 2.31 mmol, 4.50 equiv) andbis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxybutanedioate (intermediate168.1) (62 mg, 0.18 mmol, 0.35 equiv) and the resulting solution wasstirred overnight at room temperature. The resulting mixture wasconcentrated under vacuum and the crude product was purified byPrep-HPLC with methanol:water (1:10). This resulted in 140 mg (26%) ofN1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamideas a white solid. ¹H-NMR (300 MHz. DMSO, ppm): δ 7.65 (m, 4H), 7.11 (m,2H), 6.83 (m, 2H), 6.58 (m, 2H), 6.41 (m, 4H), 4.09 (m 32H), 3.45 (m,17H), 3.43 (m, 5H), 3.31 (m, 9H), 2.51 (m, 6H). MS (m/z): 1079 [M+H]⁺.

Example 174N1,N4-bis(1-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)-1-oxo-5,8,1l-trioxa-2-azatridecan-13-yl)-2,3-dihydroxysuccinamide

Intermediate 174.1,1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)urea

To 4-nitrophenyl3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylcarbamate(prepared by the procedure described in example 38) (200 mg, 0.40 mmol,1.00 equiv, 95%) in DMF (5 mL) was added TEA (170 mg, 1.60 mmol, 4.00equiv, 95%) and 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (90 mg,0.39 mmol, 1.00 equiv, 95%) and the resulting solution was stirred for 2h. The mixture was then concentrated under vacuum, diluted with 10 mL ofwater and then extracted with 3×30 mL of ethyl acetate. The organiclayers were combined, washed with 3×30 mL of brine, dried over anhydroussodium sulfate and then evaporated. The residue was applied onto asilica gel column with ethyl acetate/petroleum ether (1:5˜1:1). Thisresulted in 160 mg (72%) of1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)ureaas pale-yellow oil.

Intermediate 174.21-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)urea

Intermediate 174.2 was prepared from1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)urea(intermediate 174.1) using the procedure described to prepareintermediate 173.2. The crude product was purified by silica gelchromatography, eluting with DCM/methanol (50:1). This resulted in 230mg of1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)urea as pale-yellow oil.

Compound 174,N1,N4-bis(1-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)-2,3-dihydroxysuccinamide

Compound 174 was prepared from1-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)urea(intermediate 174.2) using the procedures described in example 172. Thecrude product (400 mg) was purified by Prep-HPLC withmethanol:acetonitrile=60:40. This resulted in 113 mg (23%) ofN1,N4-bis(1-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)-1-oxo-5,8,11-trioxa-2-azatridecan-13-yl)-2,3-dihydroxysuccinamideas a white solid. ¹H-NMR (400 MHz, DMSO, ppm): δ 8.68 (s, 2H), 7.68 (s,2H), 7.64 (t, 2H), 7.39 (s, 2H), 7.24-7.28 (m, 6H), 6.77-6.78 (m, 4H),6.23 (s, 2H), 4.47 (s, 4H), 4.23 (s, 2H), 3.76 (s, 4H), 3.42-3.69 (m,24H), 3.28-3.36 (m, 4H), 3.20-3.24 (m, 6H), 3.02 (s, 6H). MS (m/z): 583[½M+1]⁺.

Example 175N1,N2-bis(2-(2-(24-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)oxalamide

Intermediate 175.1,N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

To4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride hydrochloride (intermediate 10.6) (9 g, 20.02 mmol, 1.00 equiv,95%) in DCM (200 mL) was added 2-(2-(2-aminoethoxy)ethoxy)ethanamine(15.6 g, 105.41 mmol, 5.00 equiv) and triethylamine (4.26 g, 42.18 mmol,2.00 equiv) and the resulting solution was stirred for 3 h at roomtemperature. The reaction mixture was diluted with 100 mL of DCM andthen washed with 2×50 mL of Brine. The mixture was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was appliedonto a silica gel column with DCM/methanol (10:1). This resulted in 3 g(28%) ofN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas brown oil.

Compound 175,N1,N2-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)oxalamide

Into a 50-mL round-bottom flask, was placed a solution ofN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 175.1) (150 mg, 0.28 mmol, 2.50 equiv, 92%) in DMF (5 mL),bis(2,5-dioxopyrrolidin-1-yl) oxalate (34 mg, 0.12 mmol, 1.00 equiv) andtriethylamine (49 mg, 0.49 mmol, 4.00 equiv). The resulting solution wasstirred overnight at room temperature. The crude product was purified byPrep-HPLC with acetonitrile:water (0.05% CF3COOH) (10%-100%). Thisresulted in 97 mg (68%) of a TFA salt ofN1,N2-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)oxalamideas a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.90 (m, 4H), 7.56 (s,2H), 7.50 (m, 4H), 6.85 (s, 2H), 4.77 (m, 4H), 4.53 (d, 2H), 3.90 (m,2H), 3.88 (m, 10H), 3.58 (m, 12H), 3.31 (s, 6H), 3.12 (m, 4H). MS (m/z):530 [½M+1]⁺.

Example 176N1,N4-bis(2-(2(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 176.1,N-(2-(2-aminoethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of2-(2-aminoethoxy)ethanamine dihydrochloride (1.0 g, 5.65 mmol, 5.52equiv) in DMF (20 mL), potassium carbonate (2.0 g, 14.39 mmol, 14.05equiv) and3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 1.6) (400 mg, 1.02 mmol, 1.00 equiv). Theresulting solution was stirred overnight at room temperature at whichtime it was diluted with 100 mL of water. The resulting solution wasextracted with 3×30 mL of ethyl acetate and the organic layers werecombined and dried over sodium sulfate and concentrated under vacuum.This resulted in 60 mg (13%) ofN-(2-(2-aminoethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas a yellow solid.

Compound 176,N1,N4-bis(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution ofN-(2-(2-aminoethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 176.1) (60 mg, 0.13 mmol, 1.00 equiv) in DMF (3 mL),bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxybutanedioate (intermediate168.1) (21 mg, 0.06 mmol, 0.47 equiv) and triethylamine (50 mg, 0.49mmol, 3.77 equiv). The resulting solution was stirred overnight at roomtemperature at which time the mixture was concentrated under vacuum. Thecrude product was purified by Prep-HPLC with acetonitrile:water (0.05%CF₃COOH) (10%-100%). This resulted in 21 mg (13%) of a TFA salt ofN1,N4-bis(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethyl)-2,3-dihydroxysuccinamideas a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.92 (d, J=7.8 Hz,2H), 7.81 (m, 2H), 7.67 (t, J=7.8 Hz, 2H), 7.57 (m, 2H), 7.55 (d, J=6.9Hz, 2H), 6.85 (m, 2H), 4.78 (s, 2H), 4.77 (s, 2H), 4.54 (d, J=40.2 Hz,2H), 4.48 (s, 2H), 3.92 (m, 2H), 3.53 (m, 10H), 3.18 (s, 6H), 3.07 (t,J=5.4 Hz, 4H). MS (m/z): 517 [½M+1]⁺.

Example 177N1,N4-bis(2-(22-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)succinamide

Intermediate 177.1, bis(2,5-dioxopyrrolidin-1-yl) succinate

To succinic acid (3.0 g, 25.42 mmol, 1.00 equiv) in THF (50 mL) wasadded a solution of 1-hydroxypyrrolidine-2,5-dione (6.4 g, 55.65 mmol,2.20 equiv). This was followed by the addition of a solution of DCC(11.5 g, 55.83 mmol, 2.20 equiv) in THF (50 mL) dropwise with stirringat 0° C. The resulting solution was stirred overnight at roomtemperature. The reaction progress was monitored by LCMS. The solidswere collected by filtration and the filtrate was concentrated to givethe crude product. The resulting solids were washed with THF andethanol. This resulted in 2.4 g (27%) of bis(2,5-dioxopyrrolidin-1-yl)succinate as a white solid.

Compound 177,N1,N4-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)succinamide

Compound 177 was prepared using the procedure described in example 175,substituting (2,5-dioxopyrrolidin-1-yl) succinate (intermediate 177.1)for bis(2,5-dioxopyrrolidin-1-yl) oxalate. The crude product waspurified by Prep-HPLC with acetonitrile:water (0.05% CF₃COOH)(10%-100%). This resulted in 32.8 mg (8%) ofN1,N4-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)succinamideas a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.93-7.91 (d, J=8.1Hz, 4H), 7.57-7.56 (d, J=1.8 Hz, 2H), 7.50-7.47 (d, J=8.4 Hz, 4H), 6.86(s, 2H), 4.78-4.73 (d, J=13.5 Hz, 4H), 4.52 (m, 2H), 3.85 (m, 2H),3.59-3.47 (m, 18H), 3.15-3.09 (m, 10H), 2.49 (s, 4H). MS (m/z): 544[½M+1]⁺.

Example 1782,2′-oxybis(N-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)acetamide)

Intermediate 178.1, bis(2,5-dioxopyrrolidin-1-yl) 2,2′-oxydiacetate

Intermediate 178.1 was prepared using the procedure outlined in example177, substituting 2,2′-oxydiacetic acid for succinic acid. The crudeproduct was washed with ethyl acetate. This resulted in 1.5 g (19%) ofIntermediate 178.1 as a white solid.

Compound 178,2,2′-oxybis(N-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)acetamide)

Compound 178 was prepared using the procedure described in example 175,substituting bis(2,5-dioxopyrrolidin-1-yl) 2,2′-oxydiacetate(intermediate 178.1) for bis(2,5-dioxopyrrolidin-1-yl) oxalate. Thecrude product was purified by Prep-HPLC with acetonitrile:water (0.05%CF₃COOH) (10%-100%). This resulted in 39.1 mg (7%) of a TFA salt of2,2′-oxybis(N-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)acetamide)as a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.94-7.91 (m, 4H),7.57-7.56 (m, 2H), 7.51-7.48 (m, 4H), 6.87 (m, 2H), 4.82-4.76 (m, 4H),4.54-4.49 (m, 2H), 3.93-3.91 (s, 4H), 3.89-3.87 (m, 2H), 3.66-3.42 (m,22H), 3.17 (s, 6H), 3.13-3.09 (m, 4H). MS (m/z): 552 [½M+1].

Example 179(2R,3R)—N1,N4-bis(2-(2-(2-(3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)-3-oxopropoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 179.1, tert-butyl3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate

To triethyleneglycol (17.6 g, 117.20 mmol, 3.00 equiv) in anhydrous THF(70 mL), was added sodium (30 mg, 1.25 mmol, 0.03 equiv). Tert-butylacrylate (5.0 g, 39.01 mmol, 1.00 equiv) was added after the sodium haddissolved. The resulting solution was stirred overnight at roomtemperature and then neutralized with 1.0 N hydrogen chloride. Afterremoval of the solvent, the residue was suspended in 50 mL of brine andextracted with 3×50 mL of ethyl acetate. The combined organic layerswere washed with saturated brine and dried over anhydrous sodiumsulfate. After evaporation of the solvent, the tert-butyl3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (9.6 g) was isolatedas a colorless oil, which was used directly for the next reaction stepwithout further purification.

Intermediate 179.2, tert-butyl3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate

Into a 250-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of tert-butyl3-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)propanoate (intermediate 179.1)(9.6 g, 34.49 mmol, 1.00 equiv) in anhydrous pyridine (12 mL). Themixture was cooled to 0° C. and 4-methylbenzene-1-sulfonyl chloride (7.9g, 41.44 mmol, 1.20 equiv) was added slowly in several portions. Theresulting solution was stirred at 0° C. for 1-2 h and then the flaskcontaining the reaction mixture was sealed and placed in a refrigeratorat 0° C. overnight. The mixture was poured into 120 mL of water/ice, andthe aqueous layer was extracted with 3×50 mL of DCM. The combinedorganic layers were washed with 2×50 mL of cold 1.0 N hydrogen chlorideand saturated brine and dried over anhydrous sodium sulfate. The solventwas removed under vacuum to yield 13.4 g (90%) of tert-butyl3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate as pale yellow oil.

Intermediate 179.3, tert-butyl3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanoate

Into a 250-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of tert-butyl3-(2-(2-(2-(tosyloxy)ethoxy)ethoxy)ethoxy)propanoate (13.4 g, 30.98mmol, 1.00 equiv) in anhydrous DMF (100 mL) followed by potassiumphthalimide (7.5 g, 40.49 mmol, 1.31 equiv). The resulting solution washeated to 100° C. and stirred for 3 h. The reaction progress wasmonitored by LCMS. The DMF was removed under vacuum to afford a brownoil residue. To the residue was added 200 mL water and the mixture wasextracted with 3×50 mL of ethyl acetate. The combined organic layerswere washed with saturated brine and dried over anhydrous sodiumsulfate. After evaporation of solvent, The residue was applied onto asilica gel column with ethyl acetate/petroleum ether (0˜1:3). Thesolvent was removed from fractions containing phthalimide and theresidue was washed with 20% ethyl acetate/petroleum ether to yield 10.1g (78%) of tert-butyl3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanoate aspale yellow oil.

Intermediate 179.4,3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanoic acid

Into a 10-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of tert-butyl3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanoate(intermediate 179.3) (1.5 g, 3.68 mmol, 1.00 equiv) in neat2,2,2-trifluoroacetic acid (TFA; 2.0 mL). The resulting solution wasstirred for 40 min at ambient temperature. Excess TFA was removed undervacuum to afford a pale-yellow oil residue which was purified on asilica gel column eluting with ethyl acetate/petroleum ether(1:5˜1:2˜2:1) to yield 1.1 g (84%) of3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanoic acidas a white solid.

Intermediate 179.5,3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanoylchloride

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanoic acid(700 mg, 1.99 mmol, 1.00 equiv) in anhydrous DCM (30.0 mL), then oxalyldichloride (0.7 mL) was added dropwise at room temperature. Two drops ofanhydrous DMF were then added. The resulting solution was heated toreflux for 40 min. The solvent was removed under vacuum to yield 750 mgof 3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanoylchloride as pale yellow oil, which was used directly for the nextreaction step without further purification.

Intermediate 179.6,N-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)-3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanamide

To3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenamine(intermediate 31.5) (600.0 mg, 1.95 mmol, 1.00 equiv) in anhydrous DCM(5.0 mL) was added N-ethyl-N,N-diisopropylamine (DIEA; 0.5 mL). Then asolution of3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanoylchloride (intermediate 179.5) (794 mg, 2.15 mmol, 1.10 equiv) was addeddropwise with stirring at room temperature. The resulting solution wasstirred for 2 h at ambient temperature and then concentrated undervacuum. The residue was applied onto a silica gel column withDCM/methanol (100˜50:1). This resulted in 870 mg (66%) ofN-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)-3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanamideas a pale yellow syrup. The other fractions was collected and evaporatedto get an additional 200 mg of impure product.

Intermediate 179.7,3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)propanamide

Into a 100-mL round-bottom flask, was placedN-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)-3-(2-(2-(2-(1,3-dioxoisoindolin-2-yl)ethoxy)ethoxy)ethoxy)propanamide(870.0 mg, 1.36 mmol, 1.00 equiv) and 1M hydrazine monohydrate inethanol (30.0 mL, 30.0 mmol). The resulting solution was heated atreflux for 1 hour. The resulting mixture was cooled to room temperatureand concentrated under vacuum. The residual solution was diluted with 30mL of water and then extracted with 3×50 mL of DCM. The combined organiclayers were washed with brine, dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was applied onto a silica gelcolumn with DCM/methanol (100˜50:1˜10:1˜1:1). This resulted in 600 mg(85%) of3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)propanamideas a pale yellow syrup.

Compound 179,(2R,3R)—N1,N4-bis(2-(2-(2-(3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)-3-oxopropoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

To3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-N-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenyl)propanamide(intermediate 179.7) (270 mg, 0.53 mmol, 2.00 equiv) in anhydrous DMF(5.0 mL) was added (2R,3R)-bis(2,5-dioxopyrrolidin-1-yl)2,3-dihydroxysuccinate (prepared from (2R,3R)-tartaric acid as describedin example 168) (91.0 mg, 0.26 mmol, 1.00 equiv) and triethylamine (0.3mL) and the resulting solution was stirred for 2 h at 35° C. Theresulting mixture was then concentrated under vacuum. The residue waspurified by Prep-HPLC, to give 170 mg (56%) of a TFA salt of(2R,3R)—N1,N4-bis(2-(2-(2-(3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylamino)-3-oxopropoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamideas an off-white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.92 (s, 1H),7.65 (s, 2H), 7.54 (d, J=1.5 Hz, 2H), 7.36-7.46 (m, 4H), 7.02 (dd,J=7.5, 1.2 Hz, 2H), 6.90 (s, 2H), 4.83-4.75 (m, 2H), 4.65-4.60 (m, 2H),4.53 (s, 1H), 4.46 (m, 3H), 3.88-3.80 (m, 6H), 3.64-3.51 (m, 22H),3.41-3.35 (m, 4H), 3.16 (s, 6H), 2.64 (t, J==6.0 Hz, 4H). MS (m/z): 1136[M+H]⁺.

Example 180N1,N2-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)oxalamide

Compound 180,N1,N2-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)-ethyl)oxalamide

Compound 180 was prepared from compound 28 following the procedureoutlined in example 175. The crude product (400 mg) was purified byFlash-Prep-HPLC with the following conditions: Column, C18 silica gel;mobile phase, CH₃CN/H₂O/CF₃COOH=39/100/0.05 increasing toCH₃CN/H₂O/CF₃COOH=39/100/0.05 within min; Detector, UV 254 nm. Thisresulted in 113.4 mg (11%) of a TFA salt ofN1,N2-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)oxalamideas a white solid. ¹H-NMR (300 MHz, DMSO+DCl, ppm): δ 7.766 (d, J=7.5 Hz,2H), 7.683 (s, 2H), 7.586-7.637 (m, 4H), 7.537 (d, J=7.8 Hz, 2H), 6.644(s, 2H), 4.834-4.889 (m, 2H), 4.598 (d, J=16.2 Hz, 2H), 4.446 (d, J=15.0Hz, 2H), 3.602-3.763 (m, 4H), 3.299-3.436 (m, 24H), 3.224-3.263 (m, 4H),2.975 (s, 6H), 2.825-2.863 (m, 4H). MS (m/z): 574 [M/2+H]⁺.

Example 181N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)succinamide

Compound 181,N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)succinamide

Compound 181 was prepared from compound 28 and(2,5-dioxopyrrolidin-1-yl) succinate following the procedure outlined inexample 175. The crude product (200 mg) was purified by Flash-Prep-HPLCwith the following conditions: Column, C18 silica gel; mobile phase,CH₃CN/H₂O/CF₃COOH=0.05/100/0.05 increasing toCH₃CN/H₂O/CF₃COOH=90/100/0.05 within 19 min: Detector, UV 254 nm. Thisresulted in 201 mg (78%) of a TFA salt ofN1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)succinamideas a white solid. ¹H-NMR (300 MHz, DMSO+DCl, ppm): δ 7.76 (d, J=7.5 Hz,2H), 7.68 (s, 2H), 7.63˜7.52 (m, 6H), 6.64 (s, 1H), 4.88˜4.82 (m, 2H),4.62˜4.42 (m, 4H), 3.76˜3.60 (m, 4H), 3.43˜3.30 (m, 25H), 3.14˜3.10 (m,4H), 2.97 (s, 6H), 2.86˜2.82 (m, 4H), 2.27 (s, 4H). MS (m/z): 589[M/2+1]⁺.

Example 182N1,N3-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,2-dimethylmalonamide

Compound 182,N1,N3-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,2-dimethylmalonamide

Compound 182 was prepared from compound 28 andbis(2,5-dioxopyrrolidin-1-yl) 2,2-dimethylmalonate (prepared using themethods outlined in example 168) following the procedure outlined inexample 175. The crude product (250 mg) was purified by Prep-HPLC withthe following conditions: Column, C18 silica gel; mobile phase,MeCN/H₂O/CF₃COOH=39/100/0.05, Detector, UV 254 nm. This resulted in152.3 mg (47%) of a TFA salt ofN1,N3-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,2-dimethylmalonamideas a white solid. ¹H-NMR (300 MHz, CDCl₃, ppm): δ 7.92˜7.89 (d, J=8.1Hz, 2H), 7.79 (s, 2H), 7.69˜7.64 (m, 2H), 7.57˜7.55 (d, J=7.5 Hz, 4H),3.68 (s, 2H), 4.87˜4.75 (m, 4H), 4.54˜4.49 (m, 2H), 3.90˜3.88 (m, 2H),3.67˜3.45 (m, 20H), 3.39˜3.32 (m, 4H), 3.31 (s, 6H), 3.17˜3.05 (m, 4H),1.41 (s, 1H). MS (m/z): 1189 [M+H]⁺.

Example 183N1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,2-dimethylmalonamide

Example 183,N1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,2-dimethylmalonamide

Compound 183 was prepared from intermediate 175.1 andbis(2,5-dioxopyrrolidin-1-yl) 2,2-dimethylmalonate (prepared using themethods outlined in example 168) following the procedure outlined inexample 175. The crude product was purified by Prep-HPLC withacetonitrile:water (0.05% CF₃COOH)(10%-100%). This resulted in 29.5 mg(5%) of a TFA salt ofN1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,2-dimethylmalonamideas a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.94-7.92 (m, 4H),7.57 (m, 2H), 7.51-7.49 (m 4H), 6.87 (m, 2H), 4.83-4.74 (m, 4H),4.55-4.50 (m, 2H), 3.92-3.87 (m, 2H), 3.67-3.48 (m, 8H), 3.40-3.38 (m,4H), 3.18 (s, 6H), 3.14-3.00 (m, 4H), 1.41 (s, 6H). MS (m/z): 551[½M+H]⁺.

Example 184N,N′-(2,2′-(2,2′-(2,2′-(2,2′-(pyridine-2,6-diylbis(oxy))bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,l-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Intermediate 184.1, 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl4-methylbenzenesulfonate

Into a 250-mL round-bottom flask was placed a solution of tetraethyleneglycol (50 g, 257.47 mmol, 9.81 equiv) in DCM (150 mL) and triethylamine(8 g, 79.05 mmol, 3.01 equiv). This was followed by the addition of asolution of 4-methylbenzene-1-sulfonyl chloride (5.0 g, 26.23 mmol, 1.00equiv) in DCM (10 mL) dropwise with stirring at 0° C. The resultingsolution was stirred for 2 h at room temperature, at which time it wasdiluted with 200 ml of hydrogen chloride (3N aq.). The resultingsolution was extracted with 2×150 mL of DCM and the combined organiclayers were washed with 3×150 mL of saturated sodium bicarbonate. Themixture was dried over sodium sulfate and concentrated under vacuum. Theresidue was applied onto a silica gel column with ethylacetate/petroleum ether (1:5˜ethyl acetate). This resulted in 7.0 g(77%) of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl4-methylbenzenesulfonate as colorless oil.

Intermediate 184.2, 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanol

To intermediate 184.1 (2.0 g, 5.74 mmol, 1.00 equiv) in DMF (40 mL) wasadded sodium azide (700 mg, 10.77 mmol, 1.88 equiv) and sodiumbicarbonate (800 mg, 9.52 mmol, 1.66 equiv). The resulting solution wasstirred for 2 h at 80° C. at which time the mixture was concentratedunder vacuum. The residue was diluted with 100 mL of water and thenextracted with 3×100 mL of DCM. The organic layers were combined andconcentrated under vacuum to afford 1.3 g of2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanol as light yellow oil.

Intermediate 184.3,2,6-bis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)pyridine

Into a 50-mL round-bottom flask, was placed a solution of intermediate184.2 (220 mg, 1.00 mmol, 2.38 equiv) in DMF (10 mL) and sodium hydride(40 mg, 1.00 mmol, 2.37 equiv, 60%). The resulting solution was stirredfor 30 min at room temperature, at which time 2,6-dibromopyridine (100mg, 0.42 mmol, 1.00 equiv) was added. The resulting solution was stirredfor an additional 2 h at 80° C., and then was concentrated under vacuum.The residue was applied onto a silica gel column with DCM/methanol(50:1-30:1). This resulted in 180 mg (83%) of2,6-bis(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)pyridine as lightyellow oil.

Intermediate 184.4,2-(2-(2-(2-(6-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethoxy)pyridin-2-yloxy)ethoxy)ethoxy)ethoxy)ethanamine

To intermediate 184.3 (180 mg, 0.35 mmol, 1.00 equiv) in THF/water (30/3mL) was added triphenylphosphine (400 mg, 1.52 mmol, 4.35 equiv) and theresulting solution was stirred overnight at 40° C. Aftercooling to roomtemperature, the reaction mixture was extracted with 4×50 mL of DCM andthe organic layers combined and dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was applied onto a silica gelcolumn with DCM/methanol (80:1˜20:1). This resulted in 100 mg (62%) of2-(2-(2-(2-(6-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethoxy)pyridin-2-yloxy)ethoxy)ethoxy)ethoxy)ethanamineas light yellow oil.

Compound 184,N,N′-(2,2′-(2,2′-(2,2′-(2,2′-(pyridine-2,6-diylbis(oxy))bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

To intermediate 184.4 (100 mg, 0.22 mmol, 1.00 equiv) in DCM (50 mL) wasadded triethylamine (70 mg, 0.69 mmol, 3.20 equiv) and3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (350 mg, 0.90 mmol, 4.13 equiv). The resulting solution wasstirred overnight at room temperature, and then concentrated undervacuum. The residue was purified by Prep-HPLC with CH₃CN:H₂O (0.05%CF₃COOH)=35%-40%. This resulted in 88.4 mg (29o) of a TFA salt of thetitle compound as a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ7.91-7.88 (d, 2H), 7.78 (s, 2H), 7.67-7.50 (m, 7H), 6.86 (s, 2H),6.34-6.31 (d, 2H), 4.90-4.75 (m, 4H), 4.52-4.46 (m, 2H), 4.42-4.39 (t,4H), 3.90-3.81 (m, 6H), 3.71-3.43 (m, 22H), 3.16 (s, 6H), 3.07-3.03 (t,4H). MS (m/z): 1170 [M+H]⁺

Example 1852,2′-(methylazanediyl)bis(N-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)acetamide)tris(2,2,2-trifluoroacetate)

Intermediate 185.1, bis(2,5-dioxopyrrolidin-1-yl)2,2′-(methylazanediyl)diacetate

To 2-[(carboxymethyl)(methyl)amino]acetic acid (2.0 g, 13.60 mmol, 1.00equiv) in THF (30 mL) was added DCC (6.2 g, 30.05 mmol, 2.21 equiv) anda solution of NHS (3.5 g, 30.41 mmol, 2.24 equiv) in THF (30 mL) and thereaction stirred at 0-10° C. for 2 h. The resulting solution was allowedto warm to room temperature and stirred for 16 h. The solids were thenfiltered out, and the resulting mixture was concentrated under vacuum.The crude product was re-crystallized from ethyl acetate/petroleum etherin the ratio of 1:10, to afford 2.0 g (21%) of the title compound as awhite solid.

Compound 185,2,2′-(methylazanediyl)bis(N-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-acetamide)tris(2,2,2-trifluoroacetate)

ToN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(150 mg, 0.30 mmol, 1.00 equiv) in DMF (3 mL) was added intermediate185.1 (106 mg, 0.15 mmol, 0.50 equiv, 48%) and triethylamine (150 mg,1.48 mmol, 4.97 equiv) and the reaction was stirred overnight. Themixture was concentrated under vacuum and the crude product was purifiedby Prep-HPLC with CH₃CN:H₂O (0.05% CF₃COOH) to afford 26.4 mg (12%) of aTFA salt of the title compound as a white solid. ¹H-NMR (300 MHz, CD₃OD,ppm): δ 7.92 (m, 4H), 7.5 (m, 2H), 7.50 (m, 4H), 6.85 (s, 2H), 4.81 (m,4H), 4.50 (m, 2H), 4.06 (s, 4H), 3.89 (m, 2H), 3.66-3.44 (m, 22H), 3.32(s, 6H), 3.15 (m, 4H), 3.01 (s, 3H). MS (m/z): 559 [(M+2H)/2]⁺

Example 1865-amino-N1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)isophthalamidetris(2,2,2-trifluoroacetate)

Intermediate 186.1, bis(2,5-dioxopyrrolidin-1-yl) 5-aminoisophthalate

Into a 50-mL 3-necked round-bottom flask, was placed a solution of5-aminoisophthalic acid (300 mg, 1.66 mmol, 1.00 equiv) in THF (5 mL)and 1-hydroxypyrrolidine-2,5-dione (420 mg, 3.65 mmol, 2.20 equiv). Thiswas followed by the addition of a solution of DCC (750 mg, 3.64 mmol,2.20 equiv) in THF (5 mL) dropwise with stirring at 0° C. The resultingsolution was stirred overnight at room temperature. The solids wereremoved by filtration and the filtrate was concentrated under vacuum.The crude product was purified by re-crystallization from ethanol. Thisresulted in 70 mg (11%) of the title compound as a light yellow solid.

Compound 186,5-amino-N1,N3-bis(2(2(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)isophthalamidetris(2,2,2-trifluoroacetate)

ToN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(100 mg, 0.20 mmol, 1.00 equiv) in DMF (5 mL) was added intermediate186.1 (44.8 mg, 0.12 mmol, 0.60 equiv) and triethylamine (60.4 mg, 0.60mmol, 3.00 equiv) and the reaction was stirred overnight. The resultingmixture was concentrated under vacuum and the crude product was purifiedby Prep-HPLC with CH₃CN:H₂O (0.05% CF₃COOH) to afford 32.4 mg (19%) of aTFA salt of the title compound as a white solid. ¹H-NMR (300 MHz, CD₃OD,ppm): δ 7.90-7.87 (d, J=8.4 Hz, 4H), 7.60-7.54 (3H, m), 7.46-7.44 (d,J=8.4 Hz, 4H), 7.34 (d, J=1.2 Hz, 2H), 6.82 (s, 2H), 4.89-4.71 (m, 4H),4.53-4.48 (d, J=16.2 Hz, 2H), 3.91-3.85 (m, 2H), 3.67-3.45 (m, 22H),3.33-3.32 (m, 6H), 3.18-3.01 (m, 4H). MS (m/z): 575 [(M+2H)/2]⁺

Example 1872,2′-oxybis(N-(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)acetamide)

Compound 187,2,2′-oxybis(N-(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)acetamide)

Into a 50-mL round-bottom flask, was placed a solution ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(compound 28) (150 mg, 0.28 mmol, 1.00 equiv) in DMF (5 mL),triethylamine (56 mg, 0.55 mmol, 2.01 equiv) andbis(2,5-dioxopyrrolidin-1-yl) 2,2′-oxydiacetate (intermediate 178.1) (44mg, 0.14 mmol, 0.49 equiv). The resulting solution was stirred overnightat room temperature, at which time the mixture was concentrated undervacuum. The crude product (150 mg) was purified by preparative HPLC withthe following conditions: Column, C18 silica gel; mobile phase,methanol/water=0.05/100 increasing to methanol/water=90/100 within 19min; Detector, UV 254 nm. This resulted in 72.4 mg (44%) of the titlecompound as a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.79 (d,J=7.2 Hz, 2H), 7.71 (s, 2H), 7.49-7.58 (m, 4H), 7.36-7.37 (m, 2H), 6.82(s, 2H), 4.39-4.44 (m, 2H), 4.06 (s, 4H), 3.80 (d, J=16.2 Hz, 2H), 3.65(d, J=16.2 Hz; 2H), 3.55-3.61 (m, 16H), 3.43-3.52 (m, 12H), 3.02-3.08(m, 6H), 2.65-2.70 (m, 2H), 2.49 (s, 6H). MS (m/z): 1190 [M+H]⁺

Example 1885-bromo-N1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)isophthalamidebis(2,2,2-trifluoroacetate)

Intermediate 188.1, 5-bromoisophthalic acid

Into a 100-mL round-bottom flask, was placed a solution of isophthalicacid (10 g, 60.24 mmol, 1.00 equiv) in 98% H₂SO₄ (60 mL). This wasfollowed by the addition of N-bromosuccinimide (12.80 g, 72.32 mmol,1.20 equiv), in portions at 60° C. in 10 min. The resulting solution wasstirred overnight at 60° C. in an oil bath. The reaction was cooled toroom temperature and then quenched by the addition of water/ice. Thesolids were collected by filtration, and washed with 2×60 mL of hexane.The solid was dried in an oven under reduced pressure. The crude productwas purified by re-crystallization from ethyl acetate to give 3 g (20%)of 5-bromoisophthalic acid as a white solid.

Intermediate 188.2, bis(2,5-dioxopyrrolidin-1-yl) 5-bromoisophthalate

Into a 100-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution of 5-bromoisophthalic acid(3 g, 11.76 mmol, 1.00 equiv, 96%) in THF (20 mL) followed by NHS (3 g,26.09 mmol, 2.20 equiv) at 0-5° C. To this was added a solution of DCC(5.6 g, 27.18 mmol, 2.20 equiv) in THF (20 mL) dropwise with stirring at0-5° C. The resulting solution was stirred overnight at roomtemperature. The solids were filtered out and the filtrate wasconcentrated under vacuum. The crude product was re-crystallized fromDCM/ethanol in the ratio of 1:10. This resulted in 4 g (75%) of thetitle compound as a white solid.

Compound 188,5-bromo-N1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)isophthalamidebis(2,2,2-trifluoroacetate

Into a 50-mL round-bottom flask purged and maintained with an inertatmosphere of nitrogen, was placed a solution ofN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 175.1) (100 mg, 0.19 mmol, 2.50 equiv, 95%) in DMF (8 mL),intermediate 188.1 (35 mg, 0.08 mmol, 1.00 equiv, 98%) and triethylamine(32 mg, 0.32 mmol, 4.00 equiv). The resulting solution was stirredovernight at room temperature and then concentrated to dryness. Thecrude product was purified by Prep-HPLC with acetonitrile:water (0.05%CF₃COOH)=30%-42%. This resulted in 86 mg (75%) of a TFA salt of thetitle compound as a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 8.26(s, 1H), 8.13 (s, 2H), 7.90 (d, J=9 Hz, 4H), 7.55 (s, 2H), 7.48 (d, J=9Hz, 4H), 6.84 (s, 2H), 4.76 (m, 4H), 4.54 (m, 2H), 3.89 (m, 2H), 3.68(m, 18H), 3.53 (m, 4H), 3.33 (s, 6H), 3.18 (m, 4H). MS (m/z): 609[(M+2H)/2]⁺

Example 189N1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2-hydroxymalonamidebis(2,2,2-trifluoroacetate)

Intermediate 189.1, bis(25-dioxopyrrolidin-1-yl) 2-hydroxymalonate

Into a 100 ml 3-necked roundbottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of 2-hydroxymalonicacid (1.6 g, 13.32 mmol, 1.00 equiv) in THF (30 mL) and DCC (6.2 g,30.05 mmol, 2.26 equiv). This was followed by the addition of a solutionof NHS (3.5 g, 30.41 mmol, 2.28 equiv) in THF (30 mL) at 0-10° C. in 2h. The resulting solution was stirred for 16 h at room temperature. Thesolids were then filtered out and the filtrate was concentrated undervacuum. The crude product was re-crystallized from ethanol to give 0.5 g(12%) of the title compound as a white solid.

Compound 189,N1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2-hydroxymalonamidebis(2,2,2-trifluoroacetate)

ToN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide (intermediate 175.1) (100 mg,0.20 mmol, 1.00 equiv) in DMF (2 mL), was added Intermediate 189.1 (29mg, 0.10 mmol, 0.45 equiv) and triethylamine (90 mg, 4.50 equiv) and thereaction was stirred for 3 h at 30° C. The mixture was concentratedunder vacuum and the crude product was purified by Prep-HPLC withacetonitrile:water (0.05% CF₃COOH) (10%-100%) to afford 36.5 mg (30%) ofa TFA salt of the title compound as a white solid. ¹H-NMR (300 MHz.CD₃OD, ppm): δ 7.94-7.91 (m, 4H), 7.57-7.56 (m, 2H), 7.51-7.48 (m, 4H),6.87 (m, 2H), 4.82-4.76 (m, 4H), 4.54-4.49 (m, 2H), 3.93-3.91 (s, 4H),3.89-3.87 (m, 2H), 3.66-3.42 (m, 22H), 3.17 (s, 6H), 3.13-3.09 (m, 4H).MS (m/z): 546 [(M+2H)/2]⁺

Example 190N1,N2-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)oxalamide

Compound 190,N1,N2-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)oxalamide

ToN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Intermediate 168.2) (200 mg, 0.40 mmol, 1.00 equiv) in DMF (2 mL) wasadded triethylamine (81 mg, 0.80 mmol, 2.01 equiv) andbis(2,5-dioxopyrrolidin-1-yl) oxalate (57 mg, 0.20 mmol, 0.50 equiv) andthe resulting solution was stirred overnight. The mixture wasconcentrated under vacuum and the crude product (200 mg) was purified byFlash-Prep-HPLC with the following conditions: Column, C18 silica gel:mobile phase, methanol/water=0.05/100 increasing tomethanol/water=90/100 within 25 min; Detector, UV 254 nm. This resultedin 72.3 mg (34%) ofN1,N2-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)oxalamideas a light yellow solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.77-7.81 (m,2H), 7.72 (s, 2H), 7.48-7.57 (m, 4H), 7.35-7.36 (m, 2H), 6.81-6.82 (m,2H), 4.39-4.43 (m, 2H), 3.79 (d, J=16.5 Hz, 2H), 3.65 (d, J=16.2 Hz,2H), 3.55-3.60 (m, 8H), 3.43-3.50 (m, 12H), 3.02-3.09 (m, 6H), 2.64-2.71(m, 2H), 2.49 (s, 6H). MS (m/z): 1059 [M+H]⁺

Example 191N1,N4-bis(2-(2-(23-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)succinamide

Compound 191,N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)succinamide

ToN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 168.2) (150 mg, 0.30 mmol, 1.00 equiv) in DMF (2 mL) wasadded triethylamine (60 mg, 0.59 mmol, 1.98 equiv) and intermediate177.1 (47 mg, 0.15 mmol, 0.50 equiv) and the resulting solution wasstirred overnight. The mixture was then concentrated under vacuum andthe crude product (150 mg) was purified by Flash-Prep-HPLC with thefollowing conditions: column, C18 silica gel; mobile phase,methanol/water=0.05/100 increasing to methanol/water=90/100 within 25min; Detector, UV 254 nm. This resulted in 53.1 mg (33%) ofN1,N4-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)succinamideas a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.77-7.80 (m, 2H),7.71 (s, 2H), 7.48-7.57 (m, 4H), 7.36-7.37 (m, 2H), 6.82 (s, 2H),4.39-4.44 (m, 2H), 3.79 (d, J=15.9 Hz, 2H), 3.66 (d, J=16.2 Hz, 2H),3.45-3.57 (m, 16H), 3.35-3.37 (m, 4H), 3.03-3.08 (m, 6H), 2.65-2.71 (m,2H), 2.49-2.50 (m, 10H). MS (m/z): 1089 [M+H]⁺

Example 1923,5-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethylcarbamoyl)benzenesulfonicacid

Intermediate 192.1, sodium3,5-bis((2,5-dioxopyrrolidin-1-yloxy)carbonyl)benzenesulfonate

To sodium 3,5-dicarboxybenzenesulfonate (1 g, 3.73 mmol, 1.00 equiv) andNHS (940 mg, 8.17 mmol, 2.20 equiv) in DMF (10 mL) at 0° C. was addeddropwise a solution of DCC (1.69 g, 8.20 mmol, 2.20 equiv) in THF (10mL) and the reaction stirred overnight. The solids were removed byfiltration and the filtrate was concentrated under vacuum to afford 500mg (29%) of the title compound as a white solid.

Compound 192,3,5-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl-carbamoyl)benzenesulfonicacid

ToN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 175.1) (100 mg, 0.20 mmol, 1.00 equiv) in DMF (2 mL) wasadded intermediate 192.1 (45 mg, 0.10 mmol, 0.50 equiv) andtriethylamine (90 mg, 4.50 equiv) and the resulting solution was stirredovernight. The mixture was concentrated under vacuum and the crudeproduct was purified by Prep-HPLC with acetonitrile:water (0.05%CF₃COOH)(10%-100%) to afford 30.6 mg (22%) of a TFA salt of the titlecompound as a white solid. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 8.35-8.34 (m,3H), 7.84-7.81 (m, 4H), 7.48 (m, 2H), 7.41-7.38 (m, 4H), 6.75 (m, 2H),4.87-4.70 (m, 4H), 4.56-4.50 (m, 2H), 3.92-3.85 (m, 2H), 3.70-3.42 (m,22H), 3.37-3.32 (m, 6H), 3.20-3.06 (m, 4H). MS (m/z): 608 [[(M+2H)/2]⁺

Example 193N1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-5-hydroxyisophthalamide

Intermediate 193.1, 5-hydroxyisophthalic acid

To dimethyl 5-hydroxyisophthalate (4.0 g, 19.03 mmol, 1.00 equiv) in THF(10 mL) was added lithium hydroxide (20 mL, 2M in water) and theresulting solution was stirred overnight at 40° C. The mixtureconcentrated under vacuum to remove the organic solvents and then the pHof the solution was adjusted to ˜2 with 6N hydrochloric acid. Theresulting solids were collected by filtration and dried in a vacuum ovento afford 2.0 g (58%) of 5-hydroxyisophthalic acid as a white solid.

Intermediate 193.2, bis(2,5-dioxopyrrolidin-1-yl) 5-hydroxyisophthalate

To 5-hydroxyisophthalic acid (Intermediate 193.1; 1 g, 5.49 mmol, 1.00equiv) and NHS (1.39 g, 2.20 equiv), in THF (5 mL) at 0° C. was addeddropwise a solution of DCC (2.4 g, 2.20 equiv) in THF (5 mL). Theresulting solution was stirred overnight at room temperature, thenfiltered and concentrated under vacuum to give 0.5 g (22%) of the titlecompound as a white solid.

Compound 193,N1,N3-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-5-hydroxyisophthalamide

ToN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 175.1) (100 mg, 0.20 mmol, 1.00 equiv) in DMF (2 mL) wasadded Intermediate 193.2 (34 mg, 0.09 mmol 0.45 equiv) and triethylamine(90 mg, 4.50 equiv) and the reaction was stirred overnight. The mixturewas concentrated under vacuum and the crude product was purified byPrep-HPLC with acetonitrile:water (0.05% CF₃COOH)(10%-100%) to afford 30mg (24%) of a TFA salt of the title compound as a white solid. ¹H-NMR(300 MHz, CD₃OD, ppm): δ 7.91-7.88 (m, 4H), 7.71-7.70 (m, 1H), 7.56-7.55(m, 2H), 7.47-7.44 (m, 4H), 7.37-7.36 (m, 2H), 6.84 (m, 2H), 4.87-4.70(m, 4H), 4.53-4.48 (m, 2H), 3.92-3.85 (m, 2H), 3.67-3.46 (m, 22H),3.37-3.32 (m, 6H), 3.17-3.07 (m, 4H). MS (m/z): 576 [[(M+2H)/2]⁺

Example 194(2R,3R)—N1,N4-bis(3-((3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)propyl)(methyl)amino)propyl)-2,3-dihydroxysuccinamide

Intermediate 194.1,N-(3-((3-aminopropyl)(methyl)amino)propyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

To a solution of N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine (560mg, 3.85 mmol) dissolved in DCM (20 mL), was added triethylamine (300mg, 2.96 mmol) and3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (300 mg, 0.77 mmol). The resulting solution was stirred for 3 hat room temperature. After removing the solvent, the resulting residuewas diluted with EtOAc (50 mL), washed with water (2×10 mL) and driedover anhydrous sodium sulfate. The crude product was purified byFlash-Prep-HPLC with H₂O:MeOH (1:4) to afford 300 mg (74%) ofN-(3-((3-aminopropyl)(methyl)amino)propyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas a yellow oil.

Compound 194,(2R,3R)—N1,N4-bis(3-((3-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)propyl)(methyl)amino)propyl)-2,3-dihydroxysuccinamide

To a solution ofN-(3-((3-aminopropyl)(methyl)amino)propyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Intermediate 194.1, 300 mg, 0.60 mmol) in DMF (2 mL) was added(2R,3R)-bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (preparedfrom (2R,3R)-tartaric acid as described in example 168) (91 mg, 0.27mmol) and triethylamine (270 mg, 2.67 mmol) and the resulting solutionwas stirred for 2 h at room temperature and the reaction progress wasmonitored by LCMS. Upon completion, the mixture was concentrated undervacuum and the crude product was purified by Prep-HPLC withacetonitrile:water (0.05% CF₃COOH) (20%-29%) to afford 30.9 mg (8%) ofthe title compound as a TFA salt. ¹H-NMR (300 MHz, CD₃OD, ppm):7.90-7.88 (m, 2H), 7.80 (m, 2H), 7.69-7.65 (m, 2H), 7.58-7.56 (m, 4H),6.85 (m, 2H), 4.87-4.71 (m, 4H), 4.54-4.44 (m, 4H), 3.88-3.82 (m, 2H),3.62-3.53 (m, 4H), 3.22 (m 6H), 3.13-3.09 (m, 6H), 3.01-2.97 (m, 4H),2.88 (m, 6H), 2.00-1.96 (m, 8H). LCMS (ES, m/z): 1114 [M+H]⁺.

Example 1952,2′-oxybis(N-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)acetamide)

Compound 195,2,2′-oxybis(N-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)acetamide)

To a solution ofN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(150 mg, 0.30 mmol) in DMF (2 mL) was added triethylamine (60 mg, 0.59mmol) and bis(2,5-dioxopyrrolidin-1-yl) 2,2′-oxydiacetate (intermediate178.1) (49 mg, 0.15 mmol) and the resulting solution was stirredovernight. After removal of the solvent, the crude product (150 mg) waspurified by Flash-Prep-HPLC (C18 silica gel; methanol/water=0.05/100increasing to methanol/water=90/100 within 25 min) to give 44.4 mg (27%)of the title compound as a TFA salt. ¹H-NMR (300 MHz, CD₃CD, ppm):7.79˜7.76 (m, 2H), 7.70 (s, 2H), 7.57-7.50 (m, 4H), 7.36 (d, J=Hz, 2H),4.89-4.41 (m, 2H), 4.06 (m, 4H), 3.81-3.62 (m, 5H), 3.59-3.42 (m, 11H),3.33-3.31 (m, 8H), 3.07-3.01 (m, 6H), 2.71-2.64 (m, 2H), 2.48 (s, 6H).LCMS (ES, m/z): 1103[M+H]⁺.

Example 196N1,N3-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,2-dimethylmalonamide

Compound 196,N1,N3-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,2-dimethylmalonamide

ToN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(150 mg, 0.30 mmol) in DMF (2 mL) was added triethylamine (60 mg, 0.59mmol) and bis(2,5-dioxopyrrolidin-1-yl) 2,2-dimethylmalonate (preparedfrom 2,2-dimethylmalonic acid as described in Example 168) (49 mg, 0.15mmol) and the resulting solution was stirred overnight. The mixture wasconcentrated and then purified by Flash-Prep-HPLC (C18 silica gel,methanol/water=0.05/100 increasing to methanol/water=90/100 within 25min) to give 75.1 mg of the title compound (46%) as a TFA salt. ¹H-NMR(300 MHz, CD₃OD, ppm): 7.80-7.77 (m, 2H), 7.71 (s, 2H), 7.57-7.48 (m4H), 7.36-7.35 (d, J=2.1 Hz, 2H), 6.81 (d, J=1.2 Hz, 2H), 4.43-4.38 (m,2H), 3.82-3.62 (m, 4H), 3.57˜3.31 (m, 18H), 3.07-3.02 (m, 6H), 2.71-2.64(m, 2H), 2.49 (s, 6H), 1.41 (s, 6H). LC-MS (ES, m/z): 1101[M+H]⁺.

Example 197N1,N2-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)oxalamide

Compound 197,N1,N2-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)-ethyl)oxalamide

To a solution ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(compound 82) (148 mg, 0.26 mmol) in DMF (5 mL) under N₂ was addedbis(2,5-dioxopyrrolidin-1-yl) oxalate (prepared from oxalic acid asdescribed in Example 168) (31 mg, 0.11 mmol) and triethylamine (44 mg,0.44 mmol) and the resulting solution was stirred overnight. The crudeproduct was purified by Prep-HPLC with CH₃CN:H₂O (0.05%CF₃COOH)(28%-35%) to afford 101.8 mg (68%) of the title compound as aTFA salt. ¹H-NMR (300 Hz, CD₃OD, ppm): 7.94 (d, J=9 Hz, 4H), 7.58 (s,2H), 7.50 (d, J=9 Hz, 4H), 6.88 (s, 2H), 4.80 (m, 4H), 4.53 (m, 2H),3.90 (m, 2H), 3.59 (m, 16H), 3.52 (m, 2H), 3.49 (m, 12H), 3.13 (s, 6H),3.09 (m, 4H). LC-MS (ES, m/z): 574 [(M+2H)/2]⁺.

Example 1982,2′-oxybis(N-(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)acetamide)

Compound 198,2,2′-oxybis(N-(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)acetamide)

To a solution ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 82) (200 mg, 0.37 mmol) in DMF (2 mL) was addedbis(2,5-dioxopyrrolidin-1-yl) 2,2′-oxydiacetate (intermediate 178.1) (60mg) and triethylamine (184 mg). The resulting solution was stirred for 2h at room temperature at which point LCMS indicated complete conversion.The mixture was concentrated under vacuum and the crude product waspurified by Prep-HPLC with acetonitrile:water (0.05% CF₃COOH)(25%-35%).This resulted in 79.6 mg (31%) of the title compound as a TFA salt.¹H-NMR (300 MHz, CD₃OD, ppm): 7.94-7.91 (m, 4H), 7.58-7.57 (m, 2H),7.51-7.48 (m, 4H), 6.88 (m, 2H), 4.82-4.74 (m, 4H), 4.52-4.47 (m, 2H),4.06 (m, 4H), 3.90 (m, 2H), 3.64-3.42 (m, 34H), 3.15-3.13 (s, 6H),3.11-3.09 (m, 4H). LC-MS (ES, m/z): 596 [(M+2H)/2]⁺.

Example 199N1,N4-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)succinamide

Compound 199,N1,N4-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)-ethyl)succinamide

To a solution ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(compound 82) (200 mg, 0.37 mmol) in dry DMF (10 mL) under N₂ was addedbis(2,5-dioxopyrrolidin-1-yl) succinate (intermediate 177.1) (57.1 mg,0.18 mmol) and triethylamine (111 mg, 1.10 mmol). The resulting solutionwas stirred for 4 h at 25° C. in an oil bath and monitored by LCMS. Theresulting mixture was concentrated under vacuum and the crude productwas purified by Prep-HPLC with acetonitrile:water (0.05%CF₃COOH)(28%-35%). This resulted in 113.8 mg (45%) of the title compoundas a TFA salt. ¹H-NMR (300 MHz, CD₃OD, ppm): 7.93-7.91 (d, J=8.1 Hz,4H), 7.58-7.57 (m, 2H), 7.50-7.48 (m, 4H), 6.87 (s, 2H), 4.88-4.74 (m,4H), 4.55-4.49 (d, J=16.2 Hz, 2H), 3.94-3.88 (m, 2H), 3.67-3.59 (m,14H), 3.55-3.45 (m, 12H), 3.35-3.09 (m, 10H), 2.48 (s, 4H). LC-MS (ES,m/z): 588 [(M+2H)/2]⁺.

Example 200 N1,N4-bis(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)succinamidebis-hydrochloride salt

Intermediate 200.1, (S orR)—N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Intermediate 175.1 (3 g) was purified by Prep-SFC with the followingconditions: Column, Chiralpak IA, 2*25 cm, 5 um: mobile phase, CO₂(50%), iso-propanol (50%); Detector, UV 254 nm This resulted in 1 g of(S orR)—N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide (intermediate 200.1) as a yellowsolid.

Compound 200, N1,N4-bis(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)succinamidebis-hydrochloride salt

To Intermediate 200.1 (280 mg, 0.56 mmol, 2.00 equiv) in DMF (10 mL) wasadded intermediate 177.1 (87 mg, 0.28 mmol, 1.00 equiv) andtriethylamine (94.3 mg, 0.93 mmol, 4.00 equiv) and the reaction wasstirred overnight. The resulting mixture was concentrated under vacuumand the crude product (300 mg) was purified by Prep-HPLC with CH₃CN:H2O(35-55%). The product was then dissolved in 15 mL of dichloromethane andgaseous hydrochloric acid was introduced for 20 minutes, then themixture was concentrated under vacuum. The crude product was washed with3×10 mL of ether to afford 222.4 mg of Compound 200 as a light yellowsolid. ¹H-NMR (400 MHz, CD₃OD, ppm): 7.94-7.92 (d, J=8 Hz, 4H),7.56-7.52 (m, 6H), 6.82 (s, 2H), 4.89-4.84 (m, 4H), 4.52-4.48 (d, J=16.4Hz, 2H), 3.91-3.90 (d, J=4 Hz, 2H), 3.62-3.48 (m, 18H), 3.39-3.32 (m,4H), 3.19-3.10 (m, 10H), 2.57-2.55 (d, J=5.2 Hz, 4H). LCMS (ES, m/z):544 [M−2HCl]/2+H⁺.

Example 201 2,2′-oxybis(N-(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)acetamide)bis-hydrochloride salt

Compound 201, 2,2′-oxybis(N-(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)acetamide)bis-hydrochloride salt

To intermediate 200.1 (500 mg, 1.00 mmol, 1.00 equiv) in DMF (3 mL) wasadded intermediate 178.1 (150 mg, 0.46 mmol, 0.45 equiv) andtriethylamine (0.4 g, 4.50 equiv) and the resulting solution was stirredfor 2 h. The crude product was purified by Prep-HPLC with CH₃CN/H₂O(0.05% TFA) (28%-34%). The product was dissolved in 15 mL ofdichloromethane and then gaseous hydrochloric acid was introduced for 20mins. The mixture was concentrated under vacuum and the crude productwas washed with 3×10 mL of ether to afford 101.1 mg (18%) of Compound201 as a white solid. ¹H-NMR (400 MHz, CD₃OD, ppm): 7.94-7.92 (m, 4H),7.57-7.51 (m, 6H), 6.84 (s, 2H), 4.88-4.70 (m, 4H), 4.50 (s, 2H), 4.08(s, 4H), 3.92-3.91 (m, 2H), 3.90-3.54 (m, 9H), 3.50-3.49 (m, 5H),3.47-3.44 (m, 8H), 3.18 (s, 6H), 3.12-3.10 (m, 4H). LCMS (ES, m/z): 552[M−2HCl]/2+H⁺.

Example 202 (S orR)—N,N′-(10,17-dioxo-3,6,21,24-tetraoxa-9,11,16,18-tetraazahexacosane-1,26-diyl)bis(3-((S)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)bis-hydrochloride salt

Intermediate 202.1, (S orR)—N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamidebis(2,2,2-trifluoroacetate)

To 2-(2-(2-aminoethoxy)ethoxy)ethanamine (30.4 g, 205.41 mmol, 8.01equiv) in dichloromethane (1000 mL) was added triethylamine (5.2 g,51.49 mmol, 2.01 equiv). This was followed by the addition of(S)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride hydrochloride (10 g, 23.42 mmol, 1.00 equiv: prepared fromintermediate 244.1 and the procedures described in Example 1) inportions at 10° C. in 1 h. The resulting solution was stirred for 15 minat room temperature. The resulting mixture was washed with 3×500 mL ofbrine, dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified by Flash-Prep-HPLC with the followingconditions: Column, C18 silica gel: mobile phase, methanol/water/TFA(4/100/0.0005) increasing to 8/10/0.0005 within 30 min: Detector, UV 254nm. This resulted in 7.2 g (42%) of intermediate 202.1 as a white solid

Compound 202, (S orR)—N,N′-(10,17-dioxo-3,6,21,24-tetraoxa-9,11,16,18-tetraazahexacosane-1,26-diyl)bis(3-((S)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)bis-hydrochloride salt

To intermediate 202.1 (500 mg, 0.69 mmol, 1.00 equiv) in DCM (10 mL) wasadded triethylamine (138 mg, 1.37 mmol, 1.99 equiv) followed by theaddition of 1,4-diisocyanatobutane (48 mg, 0.34 mmol, 0.50 equiv) inportions. The resulting solution was stirred for 10 min at roomtemperature then the crude product (500 mg) was purified byFlash-Prep-HPLC with the following conditions: Column, C18 silica gel;mobile phase, methanol/water=0.05/100 increasing to 90/100 within 30min: Detector, UV 254 nm. To the product was added 0.2 mL ofhydrochloric acid (2 N) and the solution lyophilized to afford 246.7 mg(59%) of Compound 202 as a white solid. ¹H-NMR (400 MHz, CD₃OD, ppm):7.92 (d, J=7.2 Hz, 2H), 7.83 (s, 2H), 7.69-7.65 (m, 2H), 7.60-7.55 (m,4H), 6.81 (s, 2H), 4.87-4.83 (m, 4H), 4.54-4.50 (m, 2H), 3.94-3.91 (m,2H), 3.69-3.49 (m, 18H), 3.39-3.32 (m, 4H), 3.21-3.15 (m, 10H),3.08-3.05 (m, 4H), 1.57 (s, 4H). LCMS (ES, m/z): 1145 [M−2HCl+1]⁺.

Example 203 (S orR)—N,N′-(2,2′-(2,2′-(2,2′-(1,4-phenylenebis(azanediyl))bis(oxomethylene)bis(azanediyl)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(3-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)bis-hydrochloride salt

Compound 203, (S orR)—N,N′-(2,2′-(2,2′-(2,2′-(1,4-phenylenebis(azanediyl))bis-(oxomethylene)bis(azanediyl)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(3-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)bis-hydrochloride salt

To intermediate 202.1 (400 mg, 0.55 mmol, 1.00 equiv) in DCM (10 mL) wasadded triethylamine (111 mg, 1.10 mmol, 2.00 equiv) followed by theportionwise addition of 1,4-diisocyanatobenzene (44 mg, 0.28 mmol, 0.50equiv). The resulting solution was stirred for 10 min and the crudeproduct (400 mg) was purified by Flash-Prep-HPLC with the followingconditions: Column, C18 silica gel; mobile phase, methanol/water(0.05/100) increasing to 90/100 within 30 min; Detector, UV 254 nm. Tothe product was added 0.2 mL of hydrochloric acid (2 N) and the solutionlyophilized to afford 201.7 mg (59%) of Compound 203 as a white solid.¹H-NMR (400 MHz, CD3OD, ppm): 7.84 (d, J=7.6 Hz, 2H), 7.71 (s, 2H),7.60-7.56 (m, 2H), 7.48-7.45 (m, 4H), 7.16 (s, 4H), 6.76 (s, 2H),4.70-4.66 (m, 4H), 4.42-4.38 (m, 2H), 3.78-3.74 (m, 2H), 3.53-3.48 (m,18H), 3.44-3.26 (m, 4H), 3.06-2.99 (m, 10H). LCMS (ES, m/z):1163[M−2HCl+]⁺.

Example 204N,N′-(butane-1,4-diyl)bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)acetamido)acetamido)acetamide)

Intermediate 204.1,2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)acetamido)acetamido)aceticacid

To a slurry of4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride hydrochloride (Intermediate 1.6) (283 mg, 0.66 mmol) andtriglycine (152 mg, 0.80 mmol) in THF (1.5 mL) at 0° C. was added water(1.0 mL) followed by triethylamine (202 mg, 2.0 mmol). The reaction wasallowed to warm to room temperature and stirred for 15 hours. Thesolvents were removed at reduced pressure and the residue was purifiedby preparative HPLC to give Intermediate 204.1 (122 mg) as a TFA salt.

Compound 204,N,N′-(butane-1,4-diyl)bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)acetamido)acetamido)acetamide)

Intermediate 204.1 (60 mg, 0.091 mmol) was dissolved in DMF (0.90 mL)followed by N-hydroxysuccinimide (12.6 mg, 0.11 mmol) and1,4-diaminobutane (4.0 mg, 0.045 mmol).N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (21 mg,0.11 mmol) was added and the reaction was stirred at room temperaturefor 16 hours, at which time additional 1,4-diaminobutane (1 uL) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (5 mg) wereadded. Two hours after the addition, solvent was removed at reducedpressure and the residue was purified by preparative HPLC. The titlecompound was obtained as a TFA salt (26 mg). ¹H-NMR (400 mHz, CD3OD) δ7.90 (d, j=8.6 Hz, 4H), 7.52 (d, j=1.8 Hz, 2H), 7.47 (d, j=8.6 Hz, 4H),6.84 (s, 2H), 7.75 (m, 6H), 4.44 (d, J=15.6 Hz, 2H), 3.86 (s, 4H), 3.81(s, 4H), 3.61 (s, 4H), 3.54 (m, 2H), 3.16 (m, 4H), 3.16 (s, 6H), 1.49(m, 4H). MS (m/z): 1636.98 [M+H]⁺.

Example 205N1,N4-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 205,N1,N4-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

To a solution ofN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 175.1) (110 mg, 0.22 mmol) in DMF (2.0 mL) was addedbis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (Intermediate168.1) (34 mg, 0.10 mmol) and the reaction was stirred for 10 minutes.The solvent was removed under vacuum and the residue was purified bypreparative HPLC to give the title compound (23 mg) as a TFA salt.¹H-NMR (400 mHz. CD3OD) δ 7.81 (m, 4H), 7.44 (s, 1H), 7.37 (m, 2H), 6.75(s, 1H), 4.64 (m, 4H), 4.37 (m, 4H), 3.72 (m, 2H), 3.46 (m, 10H), 3.38(m, 12H), 3.02 (m, 10H). MS (m/z): 1117.02 [M+H]⁺.

Example 206N,N′-(2,2′-(2,2′-(2,2′-(1,4-phenylenebis(methylene))bis(azanediyl)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Intermediate 206.1,N,N′-(1,4-phenylenebis(methylene))bis(2-(2-(2-aminoethoxy)ethoxy)ethanamine)

A solution of terephthalaldehyde (134 mg, 1.0 mmol) and2,2′-(ethane-1,2-diylbis(oxy))diethanamine (1.48 g, 10.0 mmol) in DCM(10 mL) was stirred at room temperature. After 15 minutes sodiumtriacetoxyborohydride (636 mg, 3.0 mmol) was added and the reaction wasstirred for 1.5 hours. Acetic acid (600 mg, 10 mmol) was then added.After stirring for an additional 1.5 hours, acetic acid (600 mg, 10mmol) and sodium triacetoxyborohydride (636 mg, 3.0 mmol) were added,and stirring was continued at room temperature. One hour later anadditional portion of sodium triacetoxyborohydride (636 mg, 3.0 mmol)was added. Twenty hours later the reaction was quenched with 1N HCl (5mL) and concentrated to dryness. Methanol (10 mL) and 12N HCl (3 drops)were added and the mixture was concentrated to dryness. The residue wasdissolved in water (10 mL) and a portion (1.0 mL) was purified bypreparative HPLC to give a TFA salt of the title compound (25 mg) as aTFA salt.

Compound 206,N,N′-(2,2′-(2,2′-(2,2′-(1,4-phenylenebis(methylene))bis(azanediyl)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

To a solution of a TFA salt of intermediate 206.1 (25 mg, 0.029 mmol) inDCM (0.5 mL) was added of4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (intermediate 1.6) (25 mg, 0.06 mmol) followed by triethylamine(24.2 mg, 0.24 mmol) and the reaction was allowed to stir at roomtemperature for 18 hours. The reaction was concentrated to dryness, andthen purified by preparative HPLC to give the title compound (8 mg) as aTFA salt. ¹H-NMR (400 mHz, CD3OD) δ 7.85 (m, 2H), 7.74 (m, 2H), 7.62 (m,6H), 7.53 (m, 4H), 6.80 (s, 1H), 4.74 (m, 6H), 4.44 (m, 2H), 4.30 (s,4H), 3.83 (m, 2H), 3.76 (m, 4H), 3.62 (m, 8H), 3.50 (m, 4H), 3.23 (m,4H), 3.10 (s, 6H), 3.02 (m, 4H). MS (m/z): 1105.05 [M+H]⁺.

Example 207(2R,3R)—N1,N4-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-23-dihydroxysuccinamide

Compound 207,(2R,3R)—N1,N4-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Following the procedures outlined in example 205, compound 207 wasprepared using (2R,3R)-bis(2,5-dioxopyrrolidin-1-yl)2,3-dihydroxysuccinate. Purification by preparative HPLC gave a TFA saltof the title compound. ¹H-NMR (400 mHz, CD3OD) δ 7.82 (m, 4H), 7.45 (m,1H), 7.38 (m, 2H), 6.75 (s, 1H), 4.64 (m, 4H), 4.37 (m, 4H), 3.74 (m,2H), 3.46 (m, 10H), 3.38 (m, 12H), 3.02 (m, 10H). MS (m/z): 1117.07[M+H]⁺.

Example 208N,N′-(13,20-dioxo-3,6,9,24,27,30-hexaoxa-12,14,19,21-tetraazadotriacontane-1,32-diyl)bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 208,N,N′-(13,20-dioxo-3,6,9,24,27,30-hexaoxa-12,14,19,21-tetraazadotriacontane-1,32-diyl)bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

To a solution of a TFA salt ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(compound 28) (47 mg, 0.061 mmol) in DMF (0.20 mL) was added1,4-diisocyanatobutane (4.0 mg, 0.03 mmol) followed bydiisopropylethylamine (15 mg, 0.12 mmol). After stirring at roomtemperature for 30 minutes, the reaction was purified by preparativeHPLC to give the title compound (31 mg) as a TFA salt. ¹H-NMR (400 mHz,CD3OD) δ 7.88 (m, 2H), 7.75 (m, 2H), 7.63 (m, 2H), 7.54 (m, 4H), 6.83(m, 2H), 4.74 (m, 4H), 4.48 (m, 2H), 3.87 (m, 2H), 3.62-3.55 (m, 14H),3.51-3.43 (m, 12H), 3.24 (m, 4H), 3.14 (s, 6H), 3.05 (m, 8H), 1.43 (m,4H). MS (m/z): 1230.99 [M+H]⁺.

Example 209N,N′-(1,1′-(1,4-phenylenebis(azanediyl))bis(1-oxo-5,8,11-trioxa-2-azatridecane-13,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 209,N,N′-(1,1′-(1,4-phenylenebis(azanediyl))bis(1-oxo-5,8,11-trioxa-2-azatridecane-13,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Following the procedures outlined in example 208, compound 209 wasprepared using 1,4-diisocyanatobenzene. Purification by preparative HPLCgave a TFA salt of the title compound. ¹H-NMR (400 mHz, CD₃OD) δ 7.78(m, 2H), 7.64 (m, 2H), 7.53 (m, 2H), 7.43 (m, 2H), 7.39 (m, 2H), 7.10(s, 4H), 6.71 (s, 2H), 4.58 (m, 4H), 4.39 (m, 2H), 3.68 (m, 2H), 3.54(s, 8H), 3.50-3.44 (m, 8H), 3.42 (m, 6H), 3.35 (m, 4H), 2.99 (s, 6H),2.95 (m, 4H). MS (m/z): 1250.98 [M+H]⁺.

Example 210(2R,3R)—N1,N4-bis(20-(4-((E)-3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)-3,6,9,12,15,18-hexaoxaicosyl)-2,3-dihydroxysuccinamide

Intermediate 210.1, (E)-ethyl3-(4-(4-(N-(20-amino-3,6,9,12,15,18-hexaoxaicosyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate

Intermediate 210.1 was prepared following the procedure outlined inExample 44.2 using 20-azido-3,6,9,12,15,18-hexaoxaicosan-1-amine. Thetitle compound was recovered in 64% yield as a yellow oil.

Intermediate 210.2,(2R,3R)—N1,N4-bis(20-(4-(4-((E)-4-(2-carboxyprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)-3,6,9,12,15,18-hexaoxaicosyl)-2,3-dihydroxysuccinamide

Intermediate 210.2 was prepared following the procedure outlined inExample 168 using (2R,3R)-bis(2,5-dioxopyrrolidin-1-yl)2,3-dihydroxysuccinate (22.4 mg, 0.065 mmol) and (E)-ethyl3-(4-(4-(N-(20-amino-3,6,9,12,15,18-hexaoxaicosyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(91.5 mg, 0.13 mmol). The title compound was recovered in 60% yield as aclear semi-solid.

Compound 210,(2R,3R)—N1,N4-bis(20-(4-(4-((E)-3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)-3,6,9,12,15,18-hexaoxaicosyl)-2,3-dihydroxysuccinamide

Compound 210 was prepared following the procedure outlined in Example 45using Intermediate 210.2 (59.6 mg). Purification by preparative HPLCgave the title compound (10 mg) as a TFA salt. ¹H-NMR (400 MHz, CD₃OD):δ7.64 (d, 4H), 7.48 (s, 1H), 7.32 (d, 4H), 7.12 (d, 4H), 3.62-3.58 (m,17H), 3.55-3.52 (m, 9H), 3.48-3.41 (m, 13H), 3.06 (s, 3H), 2.72 (s, 6H).MS (m/z): 1549.23 [M+H]⁺.

Compound 211(E)-3-(4-(4-(N-(20-amino-3,6,9,12,15,18-hexaoxaicosyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-N-(diaminomethylene)-2-methylacrylamide

Compound 211,(E)-3-(4-(4-(N-(20-amino-3,6,9,12,15,18-hexaoxaicosyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-N-(diaminomethylene)-2-methylacrylamide

Compound 211 was prepared following the procedure outlined in Example 45using (E)-ethyl3-(4-(4-(N-(20-amino-3,6,9,12,15,18-hexaoxaicosyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate(Intermediate 210.2, 13.2 mg). Purification by preparative HPLC gave thetitle compound (8.7 mg) as a TFA salt. ¹H-NMR (400 MHz, CD₃OD): δ 7.84(d, 2H), 7.52 (s, 1H), 7.35 (d, 2H), 7.12 (d, 2H), 3.74-3.70 (m, 2H),3.69-3.58 (m, 24H), 3.55-3.51 (m, 2H), 3.49-3.46 (m, 2H), 3.15-3.12 (m,2H), 3.07-3.04 (m, 2H). MS (m/z): 718.28 [M+H]⁺.

Example 212(2R,3R)—N1,N4-bis(2-(2-(2-(2-(4-(4-((E)-3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 212.1, (E)-ethyl3-(4-(4-(N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)phenoxy)-3,5-difluorophenyl)-2-methylacrylate

Compound 44.2 (100 mg, 0.175 mmol) and(2R,3R)-bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (30.1 mg,0.087 mmol) were dissolved in DMF (0.35 mL) with DIEA (67.7 mg, 0.525mmol) and stirred for 2 hours at room temperature. The solvent wasremoved and the resulting material partitioned between EtOAc (20 mL) andwater (20 mL). The organic layer was washed with saturated NaHCO₃(20mL), brine (20 mL) and dried over Na₂SO₄ to give the product (87.7 mg)as a yellow oil that was used without further purification.

Compound 212,(2R,3R)—N1,N4-bis(2-(2-(2-(2-(4-(4-((E)-3-(diaminomethyleneamino)-2-methyl-3-oxoprop-1-enyl)-2,6-difluorophenoxy)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 212 was prepared following the procedures outlined in Example45. Purification by preparative HPLC gave 9.6 mg of the title compoundas the TFA salt. ¹H-NMR (400 MHz, CD₃OD): δ 7.86 (d, 4H), 7.44 (s, 2H),7.31 (d, 4H), 7.11 (d, 4H), 4.44 (s, 2H), 3.61-3.53 (m, 21H), 3.50-3.41(m, 15H), 3.05 (t, 4H), 2.17 (s, 6H). MS (m/z): 1286.11 [M+H]⁺.

Example 2132,2′,2″-nitrilotris(N-(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)acetamide)

Compound 213,2,2′,2-nitrilotris(N-(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)-ethyl)acetamide)

Compound 213 was prepared following the procedure outlined in Example168 using tris(2,5-dioxopyrrolidin-1-yl) 2,2′,2″-nitrilotriacetate (75mg, 0.156 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 254 mg, 0.467 mmol). Purification by preparative HPLC gavethe title compound (32.0 mg) as the TFA salt. ¹H-NMR (400 MHz, CD3OD): δ7.88 (d, 3H), 7.75 (s, 3H), 7.63 (t, 3H), 7.54 (t, 6H), 6.82 (s, 3H),4.84-4.75 (m, 6H), 4.48 (d, 3H), 3.86 (m, 3H), 3.85-3.37 (m, 54H), 3.14(s, 9H), 3.02 (t, 6H). MS (m/z): 1777.07 [M+H]⁺.

Example 214N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Intermediate 214.1,N-(32-azido-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

A solution of32-azido-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontan-1-amine (436.9mg, 0.777 mmol) in dry DMF (3.5 mL) under N2 was cooled to 0° C. Asolution of3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzene-1-sulfonylchloride (300 mg, 0.706 mmol) and DIEA (273.2 mg, 2.118 mmol) in DMF (3mL) was added dropwise. After 60 minutes LCMS indicated completeconversion and the solvent was removed to giveN-(32-azido-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(620 mg) as a yellow oil which was used without further purification.

Compound 214,N-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

To a solution ofN-(32-azido-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Intermediate 214.1, 620 mg, 0.706 mmol) in THF/H₂O (10:1 v/v, 14.3 mL)under N2 was added trimethylphosphine (214.8 mg, 2.82 mmol). Theresulting solution was stirred overnight at which point LCMS indicatedcomplete conversion. The solvent was removed to give 819 mg of an orangeoil, a portion of which was purified by preparative HPLC to give thetitle compound as a TFA salt. ¹H-NMR (400 MHz, CD₃OD): δ 7.90 (d, 1H),7.68 (s, 1H), 7.62 (t, 1H), 7.55 (m, 2H), 6.82 (s, 1H), 3.85 (m, 1H),3.78 (q, 3H), 3.70-3.58 (m, 55H), 3.52 (m, 2H), 3.46 (t, 3H), 3.18 (t,3H), 3.11 (s, 3H), 3.03 (t, 2H). MS (m/z): 855.24 [M+H]⁺.

Example 215N1,N3,N5-tris(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)benzene-1,3,5-tricarboxamide

Compound 215,N1,N3,N5-tris(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)benzene-1,3,5-tricarboxamide

To a solution ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 75 mg, 0.0968) in DMF (0.5 mL) was addedbenzene-1,3,5-tricarboxylic acid (6.7 mg, 0.0319 mmol), DIEA (37.5 mg,0.291 mmol), and finally HATU (40.4 mg, 0.107 mmol). The reaction wasstirred for 60 minutes at room temperature at which point LCMS indicatedcomplete conversion. The resulting solution was diluted withacetonitrile/water solution (1:1 v/v) and filtered. Purification bypreparative HPLC gave the title compound (37.7 mg) as a TFA salt. ¹H-NMR(400 MHz, CD₃OD): δ8.37 (s, 3H), 7.84 (d, 2H), 7.83 (s, 2H), 7.62 (t,2H), 7.51-7.50 (m, 4H), 6.79 (s, 2H), 4.83-4.70 (m, 5H), 4.46 (d, 2H),3.86 (q, 2H), 3.67-3.53 (m, 27H), 3.45 (t, 5H), 3.39 (t, 5H), 3.14 (s,7H), 2.98 (t, 4H). MS (m/z): 1797.15 [M+H]⁺.

Example 216N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)terephthalamide

Compound 216,N1,N4-bis(2-(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)-ethyl)terephthalamide

Compound 216 was prepared following the procedure outlined in Example215 using terephthalic acid (10.7 mg, 0.0646 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 100 mg, 0.129 mmol). Purification by preparative HPLC gavethe title compound (46.3 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ7.87 (m, 6H), 7.73 (s, 2H), 7.59 (t, 2H), 7.52-7.49 (m, 4H) m, 6.80 (s,2H), 4.77-4.69 (m, 4H), 4.49 (d, 2H), 3.587 (qs, 2H), 3.67-3.54 (m,27H), 3.45 (t, 5H), 3.40 (t, 5H), 3.13 (s, 7H), 2.99 (t, 4H). MS (m/z):1224.34 [M+H]⁺.

Example 217N1,N31-bis(32-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-4,7,10,13,16,19,22,25,28-nonaoxahentriacontane-1,31-diamide

Compound 217,N1,N31-bis(32-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-4,7,10,13,16,19,22,25,28-nonaoxahentriacontane-1,31-diamide

Compound 217 was prepared following the procedure outlined in Example168 using bis(2,5-dioxopyrrolidin-1-yl)4,7,10,13,16,19,22,25,28-nonaoxahentriacontane-1,31-dioate (69.1 mg,0.0975 mmol) andN-(32-amino-3,6,9,12,15,18,21,24,27,30-decaoxadotriacontyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 214, 166.2 mg, 0.195 mmol). Purification by preparative HPLCgave the title compound (106.3 mg) as a TFA salt. ¹H-NMR (400 MHz,CD₃OD): δ 7.88 (d, 2H), 7.76 (s, 2H), 7.66 (t, 2H), 7.56 (m, 4H), 6.86(s, 2H), 3.90 (m, 2H), 3.82 (t, 2H), 3.76 (m, 6H), 3.62-3.41 (m, 28H),3.38 (m, 6H), 3.35-3.28 (m, 56H), 3.15 (s, 6H), 3.05 (t, 4H), 2.43 (t,4H). MS (m/z): 1094.37 [(M+2H)/2]⁺.

Example 2182R,3R)—N1,N4-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 218,(2R,3R)—N1,N4-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 218 was prepared following the procedure outlined in Example168 using (2R,3R)-bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate(10.2 mg, 0.0298 mmol) andN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 168.2, 30 mg, 0.0597 mmol). Purification by preparative HPLCgave the title compound (5.1 mg) as the TFA salt. ¹H-NMR (400 MHz,CD₃OD): δ 7.92 (d, J=7.8 Hz, 2H), 7.82 (m, 2H), 7.67 (t, J=7.8 Hz, 2H),7.57 (m, 2H), 7.55 (d, J=,6.9 Hz, 2H0, 6.86 (m, 2H), 4.84 (s, 2H), 4.79(s, 2H), 4.54 (d, 2H), 4.48 (s, 2H), 3.92 (m, 2H), 3.53 (m, 22H), 3.18(s, 6H), 3.07 (t, J=5.4 Hz, 4H). MS (m/z): 1119.04 [M+H]⁺.

Example 219N1,N3-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)benzene-1,3-disulfonamide

Compound 219,N1,N3-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)benzene-1,3-disulfonamide

To a solution ofN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 50 mg, 0.0917 mmol) and DIEA (35.5 mg, 0.275 mmol) in dryDCM (0.183 mL) under N2 was added benzene-1,3-disulfonyl dichloride(12.7 mg, 0.0459 mmol) in DCM (0.183 mL). The reaction mixture wasstirred at room temperature for 60 minutes at which point LCMS indicatedcomplete conversion. The solvent was removed and the resulting residuebrought up in 4 mL ACN/H₂O solution (1:1). Filtration and purificationby preparative HPLC gave the title compound (16.6 mg) as a TFA salt.¹H-NMR (400 MHz, CD3OD): δ8.28 (s, 1H), 8.06 (d, 1H), 7.85 (d, 2H), 7.75(d, 2H), 7.70 (s, 1H), 7.63 (t, 2H), 7.53 (m, 3H), 6.82 (s, 1H), 4.52(d, 1H), 3.85 (d, 1H), 3.61-3.46 (m, 28H), 3.13 (s, 6H), 3.09-3.03 (m,7H). MS (m/z): 1294.99 [M+H]⁺.

Example 220N4,N4′-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)biphenyl-4,4′-disulfonamide

Compound 220,N4,N4′-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)-ethyl)biphenyl-4,4′-disulfonamide

Compound 220 was prepared following the procedure outlined in Example219 using biphenyl-4,4′-disulfonyl dichloride (16.1 mg, 0.0459 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 50 mg, 0.0917 mmol). Purification by preparative HPLC gavethe title compound (16.7 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ7.96 (d, 4H), 7.88-7.85 (m, 5H), 7.78 (s, 2H), 7.61 (t, 2H), 7.47 (d,2H), 6.78 (s, 2H), 4.74-4.69 (m, 3H), 4.45 (d, 2H), 3.88-3.83 (m, 2H),3.62-3.59 (m, 2H), 3.55-3.53 (m, 9H), 3.52-3.43 (m, 17H), 3.13 (s, 6H),3.11-3.03 (m 8H). MS (m/z): 1371.02 [M+H]⁺.

Example 221(14R,15R)-1-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)-14,15-dihydroxy-13-oxo-3,6,9-trioxa-12-azahexadecan-16-oicacid

Compound 221,(14R,15R)-1-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)-14,15-dihydroxy-13-oxo-3,6,9-trioxa-12-azahexadecan-16-oicacid

Compound 221 was prepared by isolating the mono-addition byproduct fromthe procedure outlined in Example 168 using(2R,3R)-bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (70.4 mg,0.205 mmol) and Compound 28 (223 mg, 0.409 mmol). Purification bypreparative HPLC gave the title compound (44.4 mg) as a TFA salt. ¹H-NMR(400 MHz, CD3OD): δ 7.89 (d, 1H), 7.81 (d, 1H), 7.63 (t, 1H), 7.55 (s,1H), 7.50 (t, 1H), 6.84 (s, 0.5H), 3.88-3.84 (m, 1H), 3.64-3.34 (m,22H), 3.14 (s, 4H), 3.07 (m, 2H). MS (m/z): 677.36 [M+H]⁺.

Example 222(2S,3S)—N1,N4-bis(2-(2-(2-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 222,(2S,3S)—N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 222 was prepared following the procedure outlined in Example215 using (2S,3S)-2,3-dihydroxysuccinic acid (15.5 mg, 0.103 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 112 mg, 0.206 mmol). Purification by preparative HPLC gavethe title compound (39.9 mg) as a TFA salt. ¹H-NMR (400 MHz, CD₃OD): δ7.87 (d, 2H), 7.77 (s, 2H), 7.63 (t, 2H), 7.54-7.50 (m, 4H), 6.82 (s,2H), 4.34 (s, 2H), 3.90-3.85 (m, 1H), 3.62-3.30 (m, 47H), 3.14 (m, 8H),3.05 (t, 4H). MS (m/z): 1206.95 [M+H]⁺.

Example 223N1,N4-bis(2-(2-(2-(2-(3-((R)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 223.1a, (R orS)—N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideand 223.1b (S orR)—N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 28.1, 4.5 g, 7.88 mmol, 1.00 equiv) was separated into itsenantiomers by chiral phase preparative Supercritical FluidChromatography (Prep-SFC) with the following conditions: Column,Chiralpak IA, 2*25 cm, 5 um; mobile phase, CO₂ (80%), methanol (20%);Detector, UV 254 nm.

This resulted in 1.61 g of (R orS)—N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas a yellow oil. ¹H-NMR (300 MHz, CD₃OD, ppm): δ 7.79 (d, J=7.5 Hz, 1H),7.711 (s, 1H), 7.49-7.58 (m, 2H), 7.36-7.37 (m, 1H), 6.83 (s, 1H),4.40-4.44 (m, 1H), 3.80 (d, J=16.2 Hz, 1H), 3.58-3.69 (m, 9H), 3.40-3.52(m, 4H), 3.33-3.38 (m, 3H), 3.03-3.09 (m, 3H), 2.66-2.72 (m, 1H), 2.50(s, 3H). MS (m/z): 572 [M+H]⁺.

This also gave 1.81 g of (S orR)—N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas yellow oil. ¹H-NMR (300 MHz. CD₃OD, ppm): δ 7.78-7.81 (m, 1H), 7.71(s, 1H), 7.49-7.58 (m, 2H), 7.36-7.37 (m 1H), 6.83 (s, 1H), 4.40-4.44(m, 1H), 3.80 (d, J=15.9 Hz, 1H), 3.57-3.70 (m, 9H), 3.44-3.53 (m, 4H),3.37-3.40 (m, 3H), 3.03-3.09 (m, 3H), 2.66-2.72 (m, 1H), 2.50 (s, 3H).MS (m/z): 572 [M+H]⁺.

Intermediate 223.2, (R orS)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Following the procedure outlined in example 170, intermediate 223.1a wasconverted to Intermediate 223.2.

Compound 223, N1,N4-bis(2-(2-(2-(2-(3-((R orS)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 223 was prepared following the procedures outlined in Example168 using (R orS)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 223.2, 239 mg, 0.439 mmol) andbis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (75.5 mg, 0.219mmol). Purification by preparative HPLC gave the title compound (135.5mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ 7.89 (d, 2H), 7.68 (s,2H), 7.63 (t, 2H), 7.54-7.52 (m, 4H), 6.83 (s, 2H), 4.83-4.75 (m, 5H),4.50-4.48 (m, 2H), 4.43 (d, 2H), 3.89-3.82 (m, 2H), 3.63-3.35 (m 34H),3.14 (s, 6H), 3.04 (t, 4H). MS (m/z): 1208.11 [M+H]⁺.

Example 224 N1,N4-bis(2-(2-(2-(2-(3-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 224.1, (S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Following the procedure outlined in example 170, intermediate 223.1b wasconverted to Intermediate 224.1.

Compound 224, N1,N4-bis(2-(2-(2-(2-(3-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 224 was prepared following the procedures outlined in Example223 using (S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 224.1, 274 mg, 0.502 mmol) andbis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (86.4 mg, 0.251mmol). Purification by preparative HPLC gave the title compound (159 mg)as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ 7.87 (d, 2H), 7.77 (s, 2H),7.63 (t, 2H), 6.54-6.51 (m, 4H), 6.83 (s, 2H), 4.84-4.75 (m, 4H),4.50-4.43 (m, 4H), 3.90-3.85 (m, 4H), 3.62-3.28 (m, 35H), 3.14 (s, 6H),3.04 (t, 4H). MS (m/z): 1207.11 [M+H]⁺.

Example 225 N1,N4-bis(2-(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 225.1a, (R orS)—N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideand intermediate 225.1b, (S orR)—N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(5 g, 8.76 mmol, 1.00 equiv) was separated into its enantiomers byPrep-SFC with the following conditions: Column, Chiralpak IA, 2*25 cm, 5um mobile phase, CO₂ (80%), ethanol (20%); Detector, UV 254 nm.

This resulted in 1.69 g of (R orS)—N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas a brown oil. ¹H-NMR (300 MHz, CD3OD, ppm): δ 7.85 (d, J=8.4 Hz, 2H),7.40 (d, J=8.1 Hz, 2H), 7.36 (s, 1H), 6.82 (s, 1H), 4.43 (t, 1H), 3.81(m, 1H), 3.67 (m, 9H), 3.48 (m, 4H), 3.33 (m, 2H), 3.01 (m, 1H), 2.71(m, 1H), 2.49 (s, 3H). MS (m/z): 572 [M+H]⁺.

Also isolated was 1.65 g of (S orR)—N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamideas brown oil. ¹H-NMR (300 MHz, CD3OD, ppm): δ 7.84 (d, J=8.4 Hz, 2H),7.43 (d, J=8.1 Hz, 2H), 7.36 (s, 1H), 6.82 (s, 1H), 4.42 (t, 1H), 3.81(m, 1H), 3.67 (m, 10H), 3.59 (m, 4H), 3.49 (m, 2H), 3.11 (m, 2H), 2.72(m, 1H), 2.49 (s, 3H). MS (m/z): 572 [M+H]⁺.

Intermediate 225.2, (S orR)—N-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,34-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Following the procedure outlined in example 170, intermediate 225.1b wasconverted to Intermediate 225.2.

Compound 225, N1,N4-bis(2-(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 225 was prepared following the procedures outlined in Example168 using(S)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 225.2, 302.4 mg, 0.555 mmol) andbis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (95.5 mg, 0.277mmol). Purification by preparative HPLC gave the title compound (97.1mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ 7.85 (d, 4H), 7.54 (s,2H), 7.46 (d, 4H), 6.84 (s, 2H), 4.88-4.72 (m, 3H), 4.43-4.42 (m, 2H),3.85-3.80 (m, 1H), 3.63-3.35 (m, 24H), 3.13 (s, 5H), 3.08 (t, 4H). MS(m/z): 1208.05 [M+H]⁺.

Example 226 N1,N4-bis(2-(2-(2-(2-(4-((R orS)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Intermediate 226.1, (R orS)—N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

Following the procedure outlined in example 170, intermediate 225.1a wasconverted to intermediate 226.1.

Compound 226, N1,N4-bis(2-(2-(2-(2-(4-((R orS)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 226 was prepared following the procedures outlined in Example168 using (R orS)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 226.1, 267.5 mg, 0.491 mmol) andbis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (84.5 mg, 0.245mmol). Purification by preparative HPLC gave the title compound (145.4mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ 7.89 (d, 5H), 7.54 (s,2H), 7.48 (d, 4H), 6.84 (s, 2H), 4.84-4.73 (m, 4H), 4.50-4.43 (d, 2H),4.18 (d, 2H), 3.85-3.80 (m, 2H), 3.64-3.40 (m, 32H), 3.13 (s, 6H), 3.08(t, 3H). MS (m/z): 1207.10 [M+H]⁺.

Example 227N1,N4-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 227,N1,N4-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 227 was prepared following the procedure outlined in Example168 using bis(2,5-dioxopyrrolidin-1-yl) 2,3-dihydroxysuccinate (49.6 mg,0.144 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 82, 157 mg, 0.288 mmol). Purification by preparative HPLC gavethe title compound (34.5 mg) as a TFA salt. ¹H-NMR (400 MHz, CD₃OD): δ7.89 (d, 4H), 7.53 (s, 2H), 7.45 (d, 4H), 6.83 (s, 2H), 4.77-4.74 (m,6H), 4.46 (d, 2H), 4.43 (t, 2H), 3.89-3.84 (m, 2H), 3.62-3.53 (m, 19H),3.49-3.41 (m, 13H), 3.14 (s, 6H), 3.08 (t, 4H). MS (m/z): 1206.94[M+H]⁺.

Example 228N1,N3-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)isophthalamide

Compound 228,N1,N3-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)-ethyl)isophthalamide

Compound 228 was prepared following the procedure outlined in Example215 using isophthalic acid (8.0 mg, 0.0484 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 75 mg, 0.0968 mmol). Purification by preparative HPLC gavethe title compound (45.6 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD):δ8.25 (s, 1H), 7.92 (d, 2H), 7.85 (d, 2H), 7.73 (s, 2H), 7.58 (t, 2H),7.49 (m, 5H), 6.81 (s, 2H), 4.83-4.71 (m, 4H), 4.49 (d, 2H), 3.87 (m,2H), 3.67-3.54 (m, 28H), 3.45 (t, 5H), 3.44 (q, 5H), 3.14 (s, 7H), 2.99(t, 4H). MS (min): 1223.19 [M+H]⁺.

Example 229(2R,3S)—N1,N4-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 229,(2R,3S)—N1,N4-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 25 mg, 0.0322 mmol) was dissolved in DMF (0.161 mL) withDIEA (12.4 mg, 0.0966 mmol) and (2R,3S)-2,3-dihydroxysuccinic acid (2.7mg, 0.0161 mmol). Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (PyBOP) (18.4 mg, 0.0354 mmol) was added and theresulting solution stirred for 60 minutes, at which point LCMS indicatedcomplete conversion. The reaction mixture was diluted to 2 mL withacetonitrile/water (1:1) and filtered. Purification by preparative HPLCgave the title compound (8.7 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD):δ 7.80 (d, 2H), 7.69 (s, 2H), 7.55 (t, 2H), 7.43 (m, 4H), 6.75 (s, 2H),4.80-4.75 (m, 3H), 4.39 (d, 2H), 4.24 (d, 2H), 3.76 (m, 2H), 3.64-3.25(m, 33H), 3.04 (s, 7H), 2.95 (t, 4H). MS (m/z): 1207.10 [M+H].

Example 230N1,N2-bis(2-(2(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)phthalamide

Compound 230,N1,N2-bis(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)-ethyl)phthalamide

Compound 230 was prepared by following the procedure outlined in Example215 using phthalic acid (8.0 mg, 0.0484 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 75 mg, 0.0968 mmol). Purification by preparative HPLC gavethe title compound (35.4 mg) as a TFA salt. ¹H-NMR (400 MHz, CD₃OD): δ7.87 (d, 2H), 7.76 (s, 2H), 7.63 (t, 2H), 7.50 (m, 8H), 6.79 (s, 2H),4.83-4.73 (m, 4H), 4.65 (d, 2H0, 3.85 (q, 2H), 3.62-3.39 (m, 36H), 3.10(s, 6H), 3.02 (t, 4H). MS (m/z): 1223.00 [M+H]⁺.

Example 231N1,N4-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)terephthalamide

Compound 231,N1,N4-bis(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-terephthalamide

Compound 231 was prepared following the procedure outlined in Example215 using terephthalic acid (11.4 mg, 0.0684 mmol) and4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)-N-(2-(2-(2-hydroxyethoxy)ethoxy)-ethyl)benzenesulfonamide(Compound 175.1, 100 mg, 0.136 mmol). Purification by preparative HPLCgave the title compound (9.8 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD):δ 7.86-7.85 (m, 9H), 7.83 (s, 2H), 7.50 (s, 1H), 7.41 (d, 4H), 6.80 (s,1H), 3.68-3.42 (m, 26H), 3.34 (m, 2H), 3.09-3.01 (m, 12H). MS (m/z):1135.07 [M+H]⁺.

Example 232N,N′-(10-oxo-3,6,14,17-tetraoxa-9,11-diazanonadecane-1,19-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 232,N,N′-(10-oxo-3,6,14,17-tetraoxa-9,11-diazanonadecane-1,19-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 175.1, 80 mg, 0.110 mmol) and DIEA (42.1 mg, 0.330 mmol) weredissolved in dry DCM (0.5 mL) under N2 and cooled to 0° C. A solution oftriphosgene (4.9 mg, 0.0165 mmol) in DCM (0.2 mL) was added dropwise andthe resulting solution was warmed to room temperature over 30 minutes.The solvent was removed: the resulting residue was brought up in 4 mL ofacetonitrile/water (1:1) solution and filtered. Purification bypreparative HPLC gave the title compound (8.5 mg) as a TFA salt. ¹H-NMR(400 MHz, CD3OD): δ 7.90 (d, 4H), 7.60 (s, 2H), 7.47 (d, 4H), 6.84 (s,2H), 3.58-3.42 (m, 24H), 3.12-3.05 (m, 17H). MS (m/z): 1031.96 [M+H]⁺.

Example 233N1,N4-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)terephthalamide

Compound 233,N1,N4-bis(2-(2-(2-(2-(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)-ethyl)terephthalamide

Compound 233 was prepared following the procedures outlined in Example215 using terephthalic acid (10.4 mg, 0.0628 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 82, 97.2 mg, 0.1255 mmol). Purification by preparative HPLCgave the title compound (38.9 mg) as a TFA salt. ¹H-NMR (400 MHz,CD3OD): δ 7.83 (m, 10H), 7.85 (s, 2H), 7.42 (d, 4H), 6.83 (s, 1H),3.66-3.55 (m, 28H), 3.46-3.39 (m, 11H), 3.12 (s, 7H), 3.04 (t, 4H). MS(m/z): 1223.14 [M+H]⁺.

Example 234N1,N4-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)terephthalamide

Compound 234,N1,N4-bis(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethyl)-terephthalamide

Compound 234 was prepared following the procedures outlined in Example215 using terephthalic acid (13.8 mg, 0.0833 mmol) andN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 168.2, 121.7 mg, 0.167 mmol). Purification by preparative HPLCgave the title compound (60.0 mg) as a TFA salt. ¹H-NMR (400 MHz,CD3OD): δ 7.88 (m, 6H), 7.72 (s, 2H), 7.61 (t, 2H), 7.51 (m, 4H), 6.80(s, 2H), 4.88-4.75 (m, 4H), 4.75 (d, 2H), 4.74 (m, 2H), 3.85-3.42 (m,25H), 3.12 (s, 6H), 2.99 (t, 4H). MS (m/z): 1135.11 [M+H]⁺.

Example 235N,N′-(10-oxo-3,6,14,17-tetraoxa-9,11-diazanonadecane-1,19-diyl)bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 235,N,N′-(10-oxo-3,6,14,17-tetraoxa-9,11-diazanonadecane-1,19-diyl)bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 235 was prepared following the procedures outlined in Example232 usingN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 168.2, 56.6 mg, 0.0775 mmol). Purification by preparative HPLCgave the title compound (25.0 mg) as a TFA salt. ¹H-NMR (400 MHz,CD3OD): δ 7.88 (d, 2H), 7.75 (s, 2H, 7.65 (t, 2H), 7.53 (m, 4H), 6.83(s, 2H), 4.89-4.68 (m, 2H), 3.88 (m, 2H), 3.62-3.43 (m, 21H), 3.30-3.27(nm, 6H), 3.11 (s, 7H), 3.03 (t, 4H). MS (m/z): 1031.07 [M+H]⁺.

Example 236N,N′-(10,17-dioxo-3,6,21,24-tetraoxa-9,11,16,18-tetraazahexacosane-1,26-diyl)bis(3-(6,8-dichlor)-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 236,N,N′-(10,17-dioxo-3,6,21,24-tetraoxa-9,11,16,18-tetraazahexacosane-1,26-diyl)bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 236 was prepared following the procedures outlined in Example208 using 1,4-diisocyanatobutane (5.24 mg, 0.0374 mmol) andN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 168.2, 54.7 mg, 0.0749 mmol). Purification by preparative HPLCgave the title compound (27.5 mg) as a TFA salt. ¹H-NMR (400 MHz;CD3OD): δ 7.88-7.86 (d, 2H), 7.75 (s, 2H), 7.63 (t, 2H), 7.55-7.51 (m,4H), 4.48 (m, 2H), 3.38-3.31 (m, 1H), 3.61-3.42 (m, 17H), 3.35-3.30 (m,4H), 3.13 (s, 6H), 3.08-3.02 (m, 7H), 1.45 (m, 2H). MS (m/z): 1145.04[M+H]⁺.

Example 237N,N′-(2,2′-(2,2′-(2,2′-(1,4-phenylenebis(azanediyl))bis(oxomethylene)bis(azanediyl)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 237,N,N′-(2,2′-(2,2′-(2,2′-(1,4-phenylenebis(azanediyl))bis(oxomethylene)bis(azanediyl)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 237 was prepared following the procedure outlined in Example208 using 1,4-diisocyanatobenzene (8.79 mg, 0.0549 mmol) andN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 168.2, 80.2 mg, 0.110 mmol). Purification by preparative HPLCgave the title compound (37.6 mg) as a TFA salt. ¹H-NMR (400 MHz,CD3OD): δ 7.88 (d, 2H), 7.73 (s, 2H), 7.61 (t, 2H), 7.52 (d, 2H), 7.48(d, 2H), 7.18 (s, 5H), 6.78 (s, 2H), 4.71-4.63 (m, 6H), 4.45-4.40 (m,2H), 3.81-3.77 (m, 2H), 3.58-3.55 (m, 6H), 3.53-3.50 (m, 14H), 3.47-3.44(m, 6H), 3.35-3.33 (m, 6H), 3.09 (s, 8H), 3.03 (t, 5H). MS (m/z):1165.06 [M+H]⁺.

Example 238N,N′-(10,17-dioxo-3,6,21,24-tetraoxa-9,11,16,18-tetraazahexacosane-1,26-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 238,N,N′-(10,17-dioxo-3,6,21,24-tetraoxa-9,11,16,18-tetraazahexacosane-1,26-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 238 was prepared following the procedure outlined in Example208 using 1,4-diisocyanatobutane (5.64 mg, 0.402 mmol) andN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 175.1, 58.8 mg, 0.805 mmol). Purification by preparative HPLCgave the title compound (13.8 mg) as a TFA salt. ¹H-NMR (400 MHz,CD₃OD): δ 7.86 (d, J=8 Hz, 2H), 7.72 (s, 2H), 7.61 (t, 2H), 7.52 (s,2H), 7.47 (d, J=7 Hz, 2H), 7.18 (s, 5H), 7.78 (s, 2H), 4.77-4.68 (m,5H), 4.48-4.40 (m, 2H), 3.35-3.28 (m, 2H), 3.56-3.51 (m, 16H), 3.45 (t,J=5 Hz, 5H), 3.35-3.32 (m, 10H), 3.09 (s, 6H), 3.03 (t, J=5 Hz, 3H). MS(m/z): 1145.01 [M+H]⁺.

Example 239N,N′-(2,2′-(2,2′-(2,2′-(1,4-phenylenebis(azanediyl))bis(ozomethylene)bis(azanediyl)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 239,N,N′-(2,2′-(2,2′-(2,2′-(1,4-phenylenebis(azanediyl))bis(oxomethylene)bis(azanediyl)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(oxy)bis(ethane-2,1-diyl))bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 239 was prepared following the procedure outlined in Example208 using 1,4-diisocyanatobenzene (12.5 mg, 0.078 mmol) andN-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 175.1, 113.9 mg, 0.156 mmol). Purification by preparative HPLCgave the title compound (48.9 mg) as a TFA salt. ¹H-NMR (400 MHz,CD₃OD): δ 7.87 (d, J=8 Hz, 4H), 7.52 (s, 2H), 7.40 (d, J=8 Hz, 4H), 7.18(s, 4H), 7.69 (s, 2H), 4.70-4.62 (m, 3H), 4.48-4.40) (m, 2H), 3.82-3.76(m, 2H), 3.58-3.43 (m, 21H), 3.35-3.30 (m, 4H), 3.11-3.06 (m, 11H). MS(m/z): 1165.12[M+H]⁺.

Example 240 (2S,3S)—N1,N4-bis(2-(2-(2-(2-(3-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 240, (2S,3S)—N1,N4-bis(2-(2-(2-(2-(3-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 240 was prepared following the procedures outlined in Example229 using (2S,3S)-2,3-dihydroxysuccinic acid (9.6 mg, 0.057 mmol) and (SorR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 224.1, 88.6 mg, 0.114 mmol). Purification by preparativeHPLC gave the title compound (24.5 mg) as a TFA salt. ¹H-NMR (400 MHz,CD3OD): δ 7.94 (t, 1H), 7.87 (d, 2H), 7.77 (s, 2H), 7.63 (t, 2H),7.53-7.50 (m, 4H), 6.82 (s, 2H), 4.479-4.45 (m, 2H), 4.44 (s, 2H),3.88-3.84 (m, 2H), 3.62-3.53 (m, 22H), 3.50-3.48 (m, 5H), 3.45-3.40 (m,9H), 3.13 (s, 6H), 3.04 (t, 4H). MS (m/z): 1208.02 [M+H]⁺.

Example 241 (2R,3R)—N1,N4-bis(2-(2-(2-(2-(3-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 241, (2R,3R)—N1,N4-bis(2-(2-(2-(2-(3-((R orS)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 241 was prepared following the procedures outlined in Example229 using (2R,3R)-2,3-dihydroxysuccinic acid (8.7 mg, 0.0519 mmol) and(S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 224.1, 80.5 mg, 0.104 mmol). Purification by preparativeHPLC gave the title compound (25.7) as a TFA salt. ¹H-NMR (400 MHz,CD₃OD): δ 7.87 (d, 3H), 7.76 (s, 2H), 7.63 (t, 2H), 7.54-7.51 (m, 4H),6.83 (s, 2H), 4.78-4.73 (m, 4H), 4.49-4.42 (m, 4H), 3.89-3.85 (m, 2H)3.62-3.53 (m, 22H), 3.51-48 (m, 5H), 3.46-3.38 (m, 9H), 3.14 (s, 6H),3.04 (t, 4H). MS (m/z): 1208.21 [M+H]⁺.

Example 242 (2S,3S)—N1,N4-bis(2-(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 242, (2S,3S)—N1,N4-bis(2(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 242 was prepared following the procedures outlined in Example229 using (2S,3S)-2,3-dihydroxysuccinic acid (6.3 mg, 0.0374 mmol) and(S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 225.2, 58.0 mg, 0.0749 mmol). Purification by preparativeHPLC gave the title compound (21.6 mg) as a TFA salt. ¹H-NMR (400 MHz,CD3OD): δ 7.85 (d, 4H), 7.54 (s, 2H), 7.45 (d, 3H), 6.84 (s, 1H),4.772-4.69 (m, 3H), 4.43 (s, 2H), 3.86-3.81 (m, 1H), 3.59-3.53 (m, 16H),3.49-3.39 (m, 11H), 3.12 (s, 5H), 3.08 (t, 4H). MS (m/z): 1208.14[M+H]⁺.

Example 243 (2R,3R)—N1,N4-bis(2-(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 243, (2R,3R)—N1,N4-bis(2-(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-2,3-dihydroxysuccinamide

Compound 243 was prepared following the procedures outlined in Example229 using (2R,3R)-2,3-dihydroxysuccinic acid (8.4 mg, 0.0.0499 mmol) and(S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 225.2, 77.3 mg, 0.0999 mmol). Purification by preparativeHPLC gave the title compound (23.4 mg) as a TFA salt. ¹H-NMR (400 MHz,CD3OD): δ 7.89 (d, 4H), 7.53 (s, 2H), 7.45 (d, 4H), 6.83 (s, 2H),4.81-4.71 (m, 4H), 4.49-4.41 (m, 4H), 3.89-3.83 (m, 2H), 3.60-3.53 (m,17H), 3.49-3.38 (m, 12H), 3.13 (s, 5H), 3.08 (t, 4H). MS (m/z): 1208.09[M+H]⁺.

Example 244 (S orR)—N,N′-(13,20-dioxo-3,6,9,24,27,30-hexaoxa-12,14,19,21-tetraazadotriacontane-1,32-diyl)bis(3-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Intermediate 244.1, (S orR)-4-(3-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline

Into a 2000-mL round-bottom flask, was placed a solution of4-(3-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline(intermediate 1.4; 20 g, 54.20 mmol, 1.00 equiv) in ethanol (500 mL).This was followed by the addition of D-(+)-dibenzoyl tartaric acid (19g, 53.07 mmol, 0.98 equiv), water (160 mL) and ethanol (1440 mL) at 45°C. The resulting solution was stirred for 30 min at 45° C. in an oilbath. After cooling to room temperature over 24 hours, the solids werecollected by filtration. The filter cake was dissolved in potassiumcarbonate (saturated.) and was extracted with 2×500 mL of ethyl acetate.The combined organic layers were washed with 2×500 mL of brine, driedover anhydrous sodium sulfate and concentrated under vacuum. This gave(S orR)-4-(3-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolineas a colorless oil.

Intermediate 224.1 (alternate synthesis), (S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide

(S orR)-4-(3-bromophenyl)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinoline(intermediate 244.1) was converted to (S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 224.1) following the procedures outlined for the racemicsubstrates in Example 1 and the reduction described in Example 170.

Compound 244, (S orR)—N,N′-(13,20-dioxo-3,6,9,24,27,30-hexaoxa-12,14,19,21-tetraazadotriacontane-1,32-diyl)bis(3-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 244 was prepared following the procedures outlined in Example208 using 1,4-diisocyanatobutane (6.5 mg, 0.0471 mmol) and (S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Intermediate 224.1, 72.9 mg, 0.0941 mmol). Purification by preparativeHPLC gave the title compound (34.9 mg) as a TFA salt. ¹H-NMR (400 MHz,CD3OD): δ 7.89 (d, 2H), 7.75 (s, 2H), 7.63 (t, 2H), 7.55-7.51 (m, 4H),6.83 (s, 2H), 4.48 (d, 2H), 3.90-3.85 (m, 2H), 3.59-3.55 (m, 17H),3.51-3.43 (m, 14H), 3.31-3.23 (m, 6H), 3.14 (s, 7H), 3.04 (m, 9H), 1.43(m, 4H). MS (m/z): 1232.99 [M+H]⁺.

Example 245 (S orR)—N,N′-(1,1′-(1,4-phenylenebis(azanediyl))bis(1-oxo-5,8,11-trioxa-2-azatridecane-13,1-diyl))bis(3-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)benzenesulfonamide)

Compound 245, (S orR)—N,N′-(1,1′-(1,4-phenylenebis(azanediyl))bis(1-oxo-5,8,1-trioxa-2-azatridecane-13,1-diyl))bis(3-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 245 was prepared following the procedures outlined in Example208 using (S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Intermediate 224.1, 79.1 mg, 0.102 mmol) and 1,4-diisocyanatobenzene(8.2 mg, 0.0511 mmol). Purification by preparative HPLC gave the titlecompound (43.2 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ 7.87 (d,2H), 7.72 (s, 2H), 7.61 (t, 2H), 7.51-7.46 (m, 4H), 7.17 (s, 4H), 6.78(s, 2H), 4.44-4.39 (m, 2H), 3.82-3.77 (m, 2H), 3.61 (s, 11H), 3.57-3.53(m, 13H), 3.49-3.48 (m, 6H), 3.44 (t, 5H), 3.35-3.29 (m, 6H), 3.09 (s,7H), 3.03 (t, 4H). MS (m/z): 1253.01 [M+H]⁺.

Compound 246 N1,N4-bis(2-(2-(2-(2-(4-((S orR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-terephthalamide

Compound 246, N1,N4-bis(2-(2-(2-(2-(4-((S orR)-6S-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)-terephthalamide

Compound 246 was prepared following the procedures outlined in Example215 using (S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Intermediate 224.1, 65.1 mg, 0.0841 mmol) and terephthalic acid (6.98mg, 0.042 mmol). Purification by preparative HPLC gave the titlecompound (19.3 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ 7.89-7.85(m, 6H), 7.52 (s, 2H), 7.43 (d, 4H), 6.81 (s, 2H), 4.73-4.66 (m, 3H),4.47-4.42 (m 1), 3.84-3.79 (m, 2H), 3.64-3.59 (m, 14H), 3.57-3.54 (m,1H), 3.46-3.39 (m, 8H), 3.12 (s, 6H), 3.03 (t, 4H). MS (m/z): 1233.04[M+H]⁺.

Example 247N1-(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)succinamide

Compound 247,N1-(2-(2-(2-(2-(3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)phenylsulfonamido)ethoxy)ethoxy)ethoxy)ethyl)succinamide

Compound 247 was prepared following the procedure outlined in Example215 using 4-amino-4-oxobutanoic acid (7.6 mg, 0.0646 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-3-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 28, 50 mg, 0.0646 mmol). Purification by preparative HPLC gavethe title compound (27.8 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ7.88 (d, 1H), 7.75 (s, 1H), 7.64 (t, 1H), 7.55 (s, 1H), 7.51 (d, 1H),6.84 (s, 1H), 4.78-4.71 (m, 2H), 4.55-4.48 (m, 1H), 3.81-3.75 (m, 1H),3.63-3.55 (m, 10H), 3.51-4.45 (m, 5H), 3.44-3.41 (m, 3H), 3.38-3.31 (m,3H), 3.13 (s, 3H), 3.07-3.02 (t, 2H), 2.48-2.43 (m, 4H). MS (m/z):645.32 [M+H]⁺.

Example 248N,N′-(13,20-dioxo-3,6,9,24,27,30-hexaoxa-2,14,19,21-tetraazadotriacontane-1,32-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 248,N,N′-(13,20-dioxo-3,6,9,24,27,30-hexaoxa-12,14,19,21-tetraazadotriacontane-1,32-diyl)bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 248 was prepared following the procedure outlined in Example208 using 1,4-diisocyanatobutane (7.64 mg, 0.545 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 82, 84.4 mg, 0.109 mmol). Purification by preparative HPLCgave the title compound (43.6 mg) as a TFA salt. ¹H-NMR (400 MHz,CD3OD): δ 7.89 (d, 4H), 7.54 (s, 2H), 7.45 (d, 4H), 6.84 (s, 2H),4.79-4.71 (m, 4H), 3.89-3.85 (dd, 2H), 3.59-3.56 (m, 17H), 3.49-3.43 (m,14H), 3.28-3.23 (m, 5H), 3.14 (s, 7H), 3.09-3.04 (m, 9H), 1.42 (s, 4H).MS (m/z): 1233.03 [M+H]⁺.

Example 249N,N′-(1,1′-(1,4-phenylenebis(azanediyl))bis(1-oxo-5,8,1-trioxa-2-azatridecane-13,1-diyl))bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 249,N,N′-(1,1′-(1,4-phenylenebis(azanediyl))bis(1-oxo-5,8,11-trioxa-2-azatridecane-13,1-diyl))bis(4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 249 was prepared following the procedure outlined in Example208 using 1,4-diisocyanatobenzene (7.95 mg, 0.0495 mmol) andN-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Compound 82, 76.7 mg, 0.099 mmol). Purification by preparative HPLCgave the title compound (39.6 mg) as a TFA salt. ¹H-NMR (400 MHz,CD₃OD): δ 7.87 (d, 4H), 7.51 (s, 2H), 7.40 (d, 4H), 7.16 (s, 4H), 6.79(s, 2H), 4.88-4.83 (m, 4H), 4.65-4.50 (m, 2H), 3.81-3.77 (m, 2H),3.61-3.59 (m, 9H), 3.58-3.54 (m 11H), 3.53-3.48 (m, 5H), 3.47-3.42 (m,5H), 3.35-3.30 (m, 4H), 3.11 (s, 6H), 3.07 (t, 4H). MS (m/z): 1253.04[M+H]⁺.

Example 250 (S orR)—N,N′-(13-oxo-3,6,9,17,20,23-hexaoxa-12,14-diazapentacosane-1,25-diyl)bis(4-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 250, (S— orR)—N,N′-(13-oxo-3,6,9,17,20,23-hexaoxa-12,14-diazapentacosane-1,25-diyl)bis(4-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 250 was prepared following the procedures outlined in Example232 using (S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(Intermediate 225.2, 75 mg, 0.0968 mmol). Purification by preparativeHPLC gave the title compound (26.0 mg) as a TFA salt. ¹H-NMR (400 MHz;CD₃OD): δ 7.88 (d, 4H), 7.54 (s, 2H), 7.45 (d, 4H), 6.84 (s, 2H),4.79-4.72 (m, 5H), 4.48-4.42 (m, 2H), 3.87-3.83 (m, 2H), 3.58-3.54 (m,17H), 3.49-3.43 (m, 15H), 3.24-3.22 (m, 6H), 3.12 (s, 6H), 3.08 (t, 4H).MS (m/z): 1118.96 [M+H]⁺.

Example 251 (S orR)—N,N′-(13,20-dioxo-3,6,9,24,27,30-hexaoxa-12,14,19,21-tetraazadotriacontane-1,32-diyl)bis(4-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 251, (S orR)—N,N′-(13,20-dioxo-3,6,9,24,27,30-hexaoxa-12,14,19,21-tetraazadotriacontane-1,32-diyl)bis(4-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 251 was prepared following the procedures outlined in Example208 using (S orR)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 225.2, 88.1 mg, 0.114 mmol) and 1,4-diisocyanatobutane(7.9 mg, 0.0569 mmol). Purification by preparative HPLC gave the titlecompound (56.1 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ 7.85 (d,4H), 7.54 (s, 2H), 7.45 (d, 4H), 6.84 (s, 2H), 4.77-4.74 (m, 4H),4.50-4.46 (m, 2H), 3.89-3.84 (m, 2H), 3.61-3.56 (m, 17H), 3.50-3.43 (m,14H), 3.26-3.23 (m, 6H), 3.14 (s, 71-H), 3.09-3.04 (m, 10H), 1.48 (s,4H). MS (m/z): 1233.01 [M+H]⁺.

Example 252 (S orR)—N,N′-(1,1′-(1,4-phenylenebis(azanediyl))bis(1-oxo-5,8,1-trioxa-2-azatridecane-13,1-diyl))bis(4-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 252, (S orR)—N,N′-(1,1′-(1,4-phenylenebis(azanediyl))bis(1-oxo-5,8,11-trioxa-2-azatridecane-13,1-diyl))bis(4-((SorR)-6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide)

Compound 252 was prepared following the procedures outlined in Example208 using(S)—N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-4-(6,8-dichloro-2-methyl-1,2,3,4-tetrahydroisoquinolin-4-yl)benzenesulfonamide(intermediate 225.2, 45.2 mg, 0.0584 mmol) and 1,4-diisocyanatobenzene(4.7 mg, 0.0292 mmol). Purification by preparative HPLC gave the titlecompound (20.7 mg) as a TFA salt. ¹H-NMR (400 MHz, CD3OD): δ 7.87 (d,4H), 7.51 (s, 2H), 7.39 (d, 4H), 7.16 (s, 4H), 6.79 (s, 2H), 4.72-4.61(m, 4H), 4.46-3.99 (m, 1H), 3.81-3.73 (m, 1H), 3.62-3.42 (m, 33H),3.35-3.33 (m, 5H), 3.09-3.06 (m, 13H). MS (m/z): 1252.95 [M+H]⁺.

Example 253 Cell-Based Assay of NHE-3 Activity (Pre-IncubationInhibition)

Rat and human NHE-3-mediated Na⁺-dependent H⁺ antiport was measuredusing a modification of the pH sensitive dye method originally reportedby Paradiso (Proc. Natl. Acad. Sci. USA. (1984) 81(23): 7436-7440).PS120 fibroblasts stably expressing human NHE3 and NHERF2 were obtainedfrom Mark Donowitz (Baltimore, Md.). Opossum kidney (OK) cells wereobtained from the ATCC and propagated per their instructions. The ratNHE-3 gene (GenBank M85300) was introduced into OK cells viaelectroporation, and cells were seeded into 96 well plates and grownovernight. Medium was aspirated from the wells then incubated for 30 minat 37° C. with NH₄Cl-HEPES buffer (20 mM NH₄Cl, 80 mM NaCl, 50 mM HEPES,5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, pH 7.4) containing 5 μM BCECF-AM.Cells were washed once with Ammonium free, Na⁺-free HEPES (100 mMcholine, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂,pH 7.4) and incubated in the same buffer for 10 minutes at roomtemperature to lower intracellular pH with 0-30 μM test compound. Afterincubation, NHE-3-mediated recovery of neutral intracellular pH wasinitiated by addition of Na-HEPES buffer containing 0.4 uM ethylisopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity thatdoes not inhibit NHE-3). Changes in intracellular pH were monitoredusing a FLIPR Tetra® (Molecular Devices, Sunnyvale, Calif.) byexcitation at λ_(ex) 439 to 505 nm, and measuring BCECF fluorescence atλ_(em) 538 nm. The initial rate of the fluorescence ratio change wasused as a measure of NHE-mediated Na⁺/H⁺ activity, and reported as thechange in fluorescence ratio per minute. Initial rates were plotted asthe average of 2 or more replicates, and pIC₅₀ values were estimatedusing GraphPad Prism.

TABLE 7 Data for example in human Preincubation assay: % inhibitionResult pIC₅₀ Range range A NHE3 pIC₅₀ < 6 NHE3 < 40% B NHE3 pIC₅₀ 6-740-70% C NHE3 pIC₅₀ > 7 >70% % Example pIC50 inhibition 202 C C 203 C C

Example 254 Cell-Based Assay of NHE-3 Activity (Persistent Inhibition)

The ability of compounds to inhibit human and rat NHE-3-mediatedNa⁺-dependent H⁺ antiport after application and washout was measuredusing a modification of the pH sensitive dye method described above.PS120 fibroblasts stably expressing human NHE3 and NHERF2 were obtainedfrom Mark Donowitz (Baltimore, Md.). Opossum kidney (OK) cells wereobtained from the ATCC and propagated per their instructions. The ratNHE-3 gene was introduced into OK cells via electroporation, and cellswere seeded into 96 well plates and grown overnight. Medium wasaspirated from the wells, cells were washed once with NaCl-HEPES buffer(100 mM NaCl, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl₂, 1 mMMgCl₂, pH 7.4), then overlayed with NaCl-HEPES buffer containing 0-30 μMtest compound. After a 60 min incubation at room temperature, the testdrug containing buffer was aspirated from the cells. Followingaspiration, cells were washed once with NaCl-HEPES buffer without drug,then incubated for 30 min at 37° C. with NH₄Cl-HEPES buffer (20 mMNH₄Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, pH7.4) containing 5 μM BCECF-AM. Cells were washed once with Ammoniumfree, Na⁺-free HEPES (100 mM choline, 50 mM HEPES, 10 mM glucose, 5 mMKCl, 2 mM CaCl₂. 1 mM MgCl₂. pH 7.4) and incubated in the same bufferfor 10 minutes at room temperature to lower intracellular pH.NHE-3-mediated recovery of neutral intracellular pH was initiated (10min after compound washout) by addition of Na-HEPES buffer. For the ratNHE3 assay, the Na-HEPES buffer contained 0.4 μM ethyl isopropylamiloride (EIPA, a selective antagonist of NHE-1 activity that does notinhibit NHE-3). Changes in intracellular pH were monitored using a FLIPRTetra® (Molecular Devices, Sunnyvale, Calif.) by excitation at λ_(ex)439 to 505 nm, and measuring BCECF fluorescence at λ_(em) 538 nm. Theinitial rate of the fluorescence ratio change was used as a measure ofNHE-mediated Na⁺/H⁺ activity, and reported as the change in fluorescenceratio per minute. Initial rates were plotted as the average of 2 or morereplicates, and pIC₅₀ values were estimated using GraphPad Prism.

TABLE 8 Data for example in human Persistence assay: % inhibition ResultpIC₅₀ Range range A NHE3 pIC₅₀ < 6 NHE3 < 40% B NHE3 pIC₅₀ 6-7 40-70% CNHE3 pIC₅₀ > 7 >70% Example pIC50 % inhibition 202 C C 203 C C

Example 255 Sustained Inhibition of Apical Acid Secretion in HumanOrganoid Monolayer Cell Cultures

Basal media (BM) consisted of advanced DMEM/F12 containing 10 mM HEPES(Invitrogen, 15630-080), 1:100 Glutamax (Invitrogen, 35050-061), and1:100 penicillin/streptomycin (Invitrogen, 15140-122). Supplementedbasal media (SBM) contained 1:100 N2 (Invitrogen, 17502-048), 1:50 B27(Invitrogen, 12587-010), 1 mM N-acetylcysteine (Sigma. A9165), and 10 nM[Leu15]-gastrin 1 (Sigma, G9145). Growth factors used included 50 ng permL mouse EGF (Peprotech, 315-09), 100 ng per mL mouse noggin (Peprotech,250-38), 500 ng per mL human R-spondin 1 (R&D, 4645-RS), 100 ng per mLmouse Wnt-3a (R&D, 1324-WN), 20 μM Y-27632 (Tocris, 1254), 10 mMnicotinamide (Sigma, N0636), 500 nM A83-01 (Tocris, 2939), 10 μMSB202190 (Tocris, 1264). Transwells were 0.4 μm pore polyester membrane24-well Transwell inserts (Corning). Cultures were incubated at 37° C.in 5% CO2.

Human ileum organoids were cultured in WENRNAS (Wnt, EGF, noggin.R-spondin1. Nicotinamide. A83-01, SB202190) and typically grown for 7-12days before being used to plate monolayer cultures. On day 0, organoidcultures embedded in Matrigel were treated with TrypLE Express to breakorganoids into small pieces and/or single cells. The cells wereresuspended to 0.5×10⁶ cells/mL in SBM containing WENRAY (Wnt, EGF,noggin, R-spondin1, A83-01, Y-27632). Following this step, 200 μL ofcell suspension was plated into the apical side of a 24-well Transwell(100.000 cells/well) and 600 μL of SBM with WENRAY was added to thebasolateral side. Ileum cells were differentiated with ENRA (EGF,noggin. R-spondin 1, A83-01) on day 3. The color of apical compartmentturns from pink or orange to yellow due to the increase in NHE3expression after differentiation.

Each human ileum monolayer culture well was washed twice with fresh SBMon the apical side on day 6 before compound dosing. All compound stockswere 10 mM dissolved in DMSO. Each compound stock was individually mixedwith fresh SBM to reach final compound concentration 1 μM and dosed onlyon the apical side of the monolayer (total volume 200 μl). DMSO at theequivalent concentration was used as the vehicle control. Duplicatewells were dosed for each compound. On day 8, apical media pH wasmeasured by pH electrode, to determine the ability of example compoundsto produce sustained inhibition of NHE3 activity in a human monolayerculture system by preventing proton secretion into the apicalcompartment. Each of the duplicate apical pH values for each examplecompound was compared to the average of the DMSO wells and expressed asa percent inhibition of apical acid secretion.

TABLE 9 % Inhibition (GI Result Segment) A <50% B 50-70% C >70% % %inhibition inhibition Example (ileum) (duodenum) 202 C C

Example 256 Increased Trans-Epithelial Resistance in Human OrganoidMonolayer Cell Cultures

Basal media (BM) consisted of advanced DMEM/F12 containing 10 mM HEPES(Invitrogen, 15630-080), 1:100 Glutamax (Invitrogen, 35050-061), and1:1(00 penicillin/streptomycin (Invitrogen, 15140-122). Supplementedbasal media (SBM) contained 1:100 N2 (Invitrogen, 17502-048), 1:50 B27(Invitrogen, 12587-010), 1 mM N-acetylcysteine (Sigma, A9165), and 10 nM[Leu15]-gastrin 1 (Sigma, G9145). Growth factors used included 50 ng permL mouse EGF (Peprotech, 315-09), 100 ng per mL mouse noggin (Peprotech,250-38), 500 ng per mL human R-spondin 1 (R&D, 4645-RS), 100 ng per mLmouse Wnt-3a (R&D, 1324-WN), 20 μM Y-27632 (Tocris, 1254), 10 mMnicotinamide (Sigma, N0636), 500 nM A83-01 (Tocris, 2939), 10 μMSB202190 (Tocris, 12(4). Transwells were 0.4 μm pore polyester membrane24-well Transwell inserts (Corning). Cultures were incubated at 37° C.in 5% CO2.

Human duodenum organoids were cultured in WENRNAS (Wnt, EGF, noggin,R-spondin1. Nicotinamide, A83-01, SB202190) and typically grown for 7-12days before being used to plate monolayer cultures. On day 0, organoidcultures embedded in Matrigel were treated with TrypLE Express to breakorganoids into small pieces and/or single cells. The cells wereresuspended to 0.5×10⁶ cells/mL in SBM containing WENRAY (Wnt. EGF,noggin, R-spondin1, A83-01, Y-27632). Following this step, 200 μL ofcell suspension was plated into the apical side of a 24-well Transwell(100,000 cells/well) and 600 μL of SBM with WENRAY was added to thebasolateral side. Duodenum cells were differentiated with ENA (EGF,noggin, A83-01) on day 3. The color of apical compartment turns frompink or orange to yellow due to the increase in NHE3 expression afterdifferentiation.

Each human duodenum monolayer culture well was washed twice with freshSBM on the apical side on day 6 or day 7 before dosing. All compoundstocks were 10 mM dissolved in DMSO. Each compound stock wasindividually mixed with fresh SBM to reach final compound concentration1 μM and dosed only on the apical side of the monolayer (total volume200 μl). DMSO at the equivalent concentration was used as the vehiclecontrol. Duplicate wells were dosed for each compound. Transepithelialelectrical resistance (TEER) was used as a quantitative technique tomeasure of tight junction permeability. TEER values were recorded(MERS00002. Millipore) before dosing and 30 mins and 1 hr after dosingfor all wells. Each of the duplicate TEER values following treatmentwere corrected for the individual well baseline TEER. Baseline correctedTEER for each example compound was compared to the average of the DMSOwells and expressed as a percent TEER of vehicle control.

TABLE 10 Result TEER (% of vehicle) A <100% B 100-130% C >130% TEER at30 TEER at 60 minutes minues Example (% of Vehicle) (% of Vehicle) 202 BB

Example 257 Inhibition of Intestinal Sodium Absorption in Mice

Urinary and fecal sodium excretion were measured to assess the abilityof selected example compounds to inhibit the absorption of sodium fromthe intestinal lumen. In addition, an assessment of the absence orpresence of diarrhea in response to compound treatment was made.Approximately eight-week old, male. CD-1 mice were purchased from Envigo(Livermore, Calif.), were housed 6 per cage and acclimated for at least48 hours before study initiation. Animals were fed Harlan Teklad GlobalTD.160470 rodent chow (Maddison, Wis.), standard laboratory rodent chowHarlan Teklad Global 2018 with the addition of 0.4% inorganicphosphorous. Animals had ad libitum access to food and water for theduration of the study and were maintained in a temperature and humiditycontrolled room on a standard light/dark cycle of 6 AM to 6 PM. Toinitiate the study, mice were weighed and then individually placed inmetabolic cages. Following a 3-day acclimation period to the metaboliccage, a 24-hr baseline collection of urine and feces was performed. Mice(n=8/group) were then dosed by oral gavage with test compound (15 mg/kg)or vehicle (3 mM HCl, 0.01% Tween80) at a dose volume of 5 mL/kg, twicedaily at 6 AM and 3 PM for 3 consecutive days. Each day, measurements ofbody weight, 24-hour food intake, water intake, urine volume and wetfecal weight were recorded, along with any observation of diarrhea.Fecal samples were dried using a lyophilizer for at least 3 days,following which dry weight was recorded and fecal fluid content wascalculated based on the difference between the wet and dry stoolweights. Fecal fluid content on day 3 of compound treatment wascalculated as a change from the vehicle group mean. For urine samples,the volumes were determined gravimetrically. Feces and urine wereanalyzed for sodium content by microwave plasma-atomic emissionspectroscopy or ion chromatography, respectively. Urine samples wereanalyzed on an ion chromatography system (Thermo Fisher ICS-3000 orICS-5000+) coupled with conductivity detectors. Chromatographicseparation of cations was performed using an IonPac CS12A (ThermoFisher) 2×250 mm analytical column with an isocratic elution using 25 mMmethanesulfonic acid. Concentrations were interpolated from a a standardcurve (prepared in 10 mM HCl) for sodium ion based on retention time andpeak area. Fecal sample analysis by Microwave Plasma Atomic EmissionSpectrometry (MP-AES). Dry fecal samples were ground into a fine powderon a homogenizer and the ground samples (400-600 mg aliquots weighed)were digested with nitric acid by microwave method (Mars 6). Thesedigested samples were diluted with 1% Nitric acid and analyzed onAgilent 4100 MP-AES. Concentrations were calculated relative to astandard curve (prepared in 1% Nitric acid) for sodium based on thesignal intensity. Sodium was detected at a wavelength of 588.995 nm.Twenty-four-hour urinary sodium excretion (mg/24-hours) was calculatedby multiplying urinary sodium concentration by 24-hour urine volume.Twenty-four-hour fecal sodium excretion (mg/24-hours) was calculated bymultiplying fecal sodium concentration by 24-hour dry fecal weight. Theurinary and fecal sodium excretion on day 3 of compound treatment werenormalized to dietary sodium intake and expressed as a percentage of thevehicle mean.

TABLE 11 Urinary Na Fecal fluid Excretion Fecal Na content (% ofExcretion (Δ from Result vehicle) (% of vehicle) vehicle) A >70% <150%<5 B 50-70% 150-200% 5-10 C <50% >200% >10 Fecal fluid Urinary Na FecalNa content Excretion Excretion (Δ from Diarrhea Example (% of vehicle)(% of vehicle) vehicle) (±) 203 C C C +

Example 258 Inhibition of Intestinal Sodium Absorption in Rats

Urinary sodium excretion and fecal form were measured to assess theability of selected example compounds to inhibit the absorption ofsodium from the intestinal lumen. Eight-week old, male, Sprague Dawleyrats were purchased from Envigo (Livermore, Calif.), were housed 2 percage and acclimated for at least 48 hours before study initiation.Animals were fed Harlan Teklad Global TD.160470 rodent chow (Maddison,Wis.), standard laboratory rodent chow Harlan Teklad Global 2018 withthe addition of 0.4% inorganic phosphorous. Animals had ad libitumaccess to food and water for the duration of the study and weremaintained in a temperature and humidity controlled room on a standardlight/dark cycle of 6 AM to 6 PM. On the day of study initiation, rats(n=5/group) were dosed by oral gavage with test compound or vehicle (3mM HCl, 0.01% Tween80) at a dose volume of 5 mL/kg. Immediately afterdose administration animals were placed in individual metabolic cages.At 13-hours post-dose, urine samples were collected and fecal form wasassessed. In addition, the weight of food consumed over the 13-hourperiod was measured and recorded. Fecal forms were scored according to acommon scale associated with increasing fecal water to the wettestobservation in the cage's collection funnel (1, normal pellet; 2, pelletadhering to sides of collection funnel due to moisture; 3, loss ofnormal pellet shape: 4, complete loss of shape with a blotting pattern;5, liquid fecal streams evident). Fecal form score (FFS) was calculatedfor each group as the median of each individual rat's FFS within thegroup and reported in Table 12. Fecal samples were dried using alyophilizer for at least 3 days, following which dry weight was recordedand fecal fluid content was calculated based on the difference betweenthe wet and dry stool weights. Fecal fluid content was calculated as achange from the vehicle group mean. For urine samples, the volumes weredetermined gravimetrically. Urine samples were analyzed on an ionchromatography system (Thermo Fisher ICS-3000 or ICS-5000+) coupled withconductivity detectors. Chromatographic separation of cations wasperformed using an IonPac CS12A (Thermo Fisher) 2×250 mm analyticalcolumn with an isocratic elution using 25 mM methanesulfonic acid.Concentrations were interpolated from a standard curve (prepared in 10mM HCl) for sodium based on retention time and peak area.Thirteen-s-hour urinary sodium excretion (mg/13-hours) was calculated bymultiplying urinary sodium concentration by 13-hour urine volume. Theurinary sodium excretion of compound treatment was normalized to dietarysodium intake and expressed as a percentage of the vehicle mean.

TABLE 12 Urinary Na (% of Vehicle, Result out/in) A >70% B 40-70% C <40%Urinary Na Dose (% of Vehicle, FFS Example (mg/kg) out/in) (1-5) 203 0.1C 3

Example 259 Inhibition of Intestinal Sodium and Phosphorous Absorptionin the Rat Balance Model

Urinary and fecal sodium excretion, along with urinary phosphorousexcretion are measured to assess the ability of selected examplecompounds to inhibit the absorption of sodium and phosphorous from theintestinal lumen. In addition, an assessment of fecal form in responseto compound treatment is made. Approximately eight-week old, male,Sprague Dawley rats are purchased from Envigo (Livermore, Calif.),housed 2 per cage and acclimated for at least 48 hours before studyinitiation. Animals are fed Harlan Teklad Global TD.160470 rodent chow(Maddison, Wis.), standard laboratory rodent chow Harlan Teklad Global2018 with the addition of 0.4% inorganic phosphorous. Animals have adlibitum access to food and water for the duration of the study and aremaintained in a temperature and humidity controlled room on a reversedlight/dark cycle of 6 PM to 6 AM. To initiate the study, rats areweighed and individually placed in metabolic cages. Following a 2-dayacclimation period to the metabolic cage, a 24-hr baseline collection ofurine and feces is performed. Rats (n=6/group) are then dosed by oralgavage with test compound or vehicle (3 mM HCl, 0.01% Tween80) at a dosevolume of 5 mL/kg, twice daily at 6 AM and 3 PM for 3 consecutive days.Each day, measurements of body weight, 24-hour food intake, waterintake, urine volume and wet fecal weight are recorded, along with anyobservation of diarrhea. Fecal samples are dried using a lyophilizer forat least 3 days, following which dry weight is recorded and fecal fluidcontent is calculated based on the difference between the wet and drystool weights. Fecal fluid content on day 3 of compound treatment iscalculated as a change from the vehicle group mean. For urine samples,the volumes are determined gravimetrically. Feces and urine are analyzedfor sodium and phosphorous content by microwave plasma-atomic emissionspectroscopy or ion chromatography, respectively. Urine samples areanalyzed on an ion chromatography system (Thermo Fisher ICS-3000 orICS-5000+) coupled with conductivity detectors. Chromatographicseparation of cations is performed using an IonPac CS12A (Thermo Fisher)2×250 mm analytical column with an isocratic elution using 25 mMmethanesulfonic acid. Chromatographic separation of anions is performedusing an IonPac AS18 (Thermo Fisher) 2×250 mm analytical column with anisocratic elution using 35 mM potassium hydroxide. Concentrations areinterpolated from a standard curve (prepared in 10 (nM HCl) for each ionbased on retention time and peak area. Fecal sample analysis byMicrowave Plasma Atomic Emission Spectrometry (MP-AES). Dry fecalsamples are ground into a fine powder on a homogenizer and the groundsamples (400-600 mg aliquots weighed) are digested with nitric acid bymicrowave method (Mars 6). These digested samples are diluted with 1%Nitric acid and analyzed on Agilent 4100 MP-AES. Concentrations areinterpolated from a standard curve (prepared in 1% Nitric acid) forsodium based on the signal intensity. Sodium is detected at a wavelengthof 588.995 nm. Twenty-four-hour urinary sodium and phosphorous excretion(mg/24-hours) is calculated by multiplying urinary sodium or phosphorousconcentration, respectively, by 24-hour urine volume. Twenty-four-hourfecal sodium excretion (mg/24-hours) is calculated by multiplying fecalsodium concentration by 24-hour dry fecal weight. The urinary and fecalsodium excretion and urinary phosphorous excretion on day 3 of compoundtreatment are normalized to dietary sodium or phosphorous intake,respectively, and expressed as a percentage of the vehicle mean.

Example 260 Restoration of Gastrointestinal Motility in Opioid InducedConstipation

Gastrointestinal transit was measured in mice treated with theperipherally acting p-opioid agonist loperamide to assess the ability ofselected example compounds to restore gastrointestinal motility in amodel of opioid induced constipation. Approximately eight-week old,female, CD1 rats were purchased from Envigo (Livermore, Calif.), werehoused 4 per cage and acclimated for at least 48 hours before studyinitiation. Animals were fed standard laboratory rodent chow HarlanTeklad Global 2018 (Maddison, Wis.). Animals had ad libitum access tofood and water for the duration of the acclimation period and weremaintained in a temperature and humidity controlled room on a standardlight/dark cycle of 6 AM to 6 PM. Following an overnight fast, with freeaccess to water, animals were dosed by oral gavage with varying doses oftest compound or vehicle (3 mM HCl, 0.01% Tween80), at a dose volume of5 mL/kg. Approximately fifteen minutes following oral dosing of testcompound or vehicle, animals were dosed by subcutaneous injection withloperamide (0.3 to 6 mg/kg) or vehicle (30:70 PG:0.9% NaCl) at a dosevolume of 5 mL/kg. Fifteen minutes later, animals were dosed orally withEvans Blue Dye (6%) at a dose volume of 100 μL, 30 minutes later,animals were euthanized by carbon dioxide inhalation, and the lengthfrom the pylorus to cecum (whole length of the small intestine) and thelength from the pylorus to the Evans Blue dye front were measured andrecorded. For an individual animal, the length travelled by the EvansBlue dye front was divided by the length of the whole small intestine,measured from the pylorus to the cecum, and multiplied by 100, toprovide the distance of the small intestine travelled by the dye as apercentage. In animals dosed orally with vehicle and injectedsubcutaneously with vehicle (vehicle/vehicle), the Evans Blue dye fronttravelled approximately 70% of the length of the small intestine in the30-minute period. In animals dosed orally with vehicle and injectedsubcutaneously with loperamide (vehicle/loperamide), the Evans Blue dyefront travelled approximately only 25% of the length of the smallintestine in the 30-minute period, indicating decreased gastrointestinalmotility in response to loperamide. The effect of example compounds onGIT motility in the presence of loperamide was calculated as the abilityto restore vehicle/vehicle transit distance from the vehicle/loperamidetransit, expressed as a percentage.

TABLE 13 % Restoration Result of Transit A <20% B 20-40% C >40% % DoseRestoration Example (mg/kg) of Transit 202 15 C

Example 260 Restoration of Gastrointestinal Motility in MultipleSclerosis

Gastrointestinal transit time is measured to assess the ability ofselected example compounds to restore gastrointestinal motility in amodel of multiple sclerosis. Multiple sclerosis (MS) patients oftenexperience constipation and other gastrointestinal manifestationsrelated to disturbed gastrointestinal motility. The ExperimentalAutoimmune Encephalomyelitis (EAE) mouse model is one of the mostfrequently used animal models for studying multiple sclerosis (MS), inwhich immunization against CNS-specific antigen results in centralnervous system inflammation. This model results in a spectrum of acute,chronic, and relapsing disease that results in varying degrees ofprogressive paralysis and gastrointestinal dysmotility.

Animals are 8-16 weeks of age at study initiation, and are fed standardlaboratory rodent chow Harlan Teklad Global 2018 (Maddison, Wis.).Animals have ad libitum access to food and water for the duration of thestudy and are maintained in a temperature and humidity controlled roomon a standard light/dark cycle of 6 AM to 6 PM. EAE is induced in femalemice by injection of a combination of antigen (MOG35-55. S.C.) incomplete Freund's adjuvant (CFA), and pertussis toxin (PTX, IP). Aftersomatic motor symptoms develop, generally 10 or more days' postimmunization. EAE mice are dosed by oral gavage with test compound atvarying doses (0.01 to 30 mg/kg) or vehicle (3 mM HCl, 0.01% Tween80) ata dose volume of 5 mL/kg. Test compound is administered for a singledose or twice daily for multiple doses. Fecal output is monitored for astandardized period of time (1-24 hours) and recorded as fecal pelletnumber, fecal mass and fecal dry weight. Whole gastrointestinal transittime is determined by oral gavage of carmine red or Evans Blue andcalculating the latency for dye to appear in the feces. Small intestinaltransit is measured by dosing carmine red or Evans Blue by oral gavageand measuring the distance of the leading edge of the dye from comparedto the whole length of the small intestine 15 minutes to two hoursfollowing oral dosing of the dye. Colonic motility is assessed bymeasuring time to extrusion of a single glass bead inserted astandardized distance into the distal colon. The effect of examplecompounds on GIT motility in EAE mice is calculated as the ability torestore transit distance to those observed in control mice from thoseobserved in EAE treated with vehicle, expressed as a percentage.

Example 261 Restoration of Gastrointestinal Motility in Parkinson'sDisease

Gastrointestinal transit time is measured to assess the ability ofselected example compounds to restore gastrointestinal motility in amodel of Parkinson's Disease. Parkinson's Disease (PD) is aneurodegenerative disorder characterized by chronic and progressivemotor impairment. PD patients also experience significant non-motorsymptoms including constipation and other gastrointestinalmanifestations related to disturbed gastrointestinal motility. Thetoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has beenwidely used to develop animal models for testing new therapies in thePD. This model results in motor changes and pathology that resemble PDand has also been reported to manifest gastrointestinal dysmotility(Scientific Reports, 2016 6:30269)

Animals are 8-16 weeks of age at study initiation, and fed standardlaboratory rodent chow Harlan Teklad Global 2018 (Maddison, Wis.).Animals have ad libitum access to food and water for the duration of thestudy and are maintained in a temperature and humidity controlled roomon a standard light/dark cycle of 6 AM to 6 PM. PD is induced in mice bymultiple, generally four, intraperitoneal injections of MPTP. After MPTPis injected, generally 4 to 20 days' post injection, PD mice are dosedby oral gavage with test compound at varying doses (0.01 to 30 mg/kg) orvehicle (3 mM HCl, 0.01% Tween80) at a dose volume of 5 mL/kg. Testcompound is administered once or twice daily for multiple doses. Fecaloutput is monitored for a standardized period of time (1-24 hours) andrecorded as fecal pellet number, fecal mass and fecal dry weight. Wholegastrointestinal transit time is determined by oral gavage of carminered or Evans Blue and calculating the latency for dye to appear in thefeces. Small intestinal transit is measured by dosing carmine red orEvans Blue by oral gavage and measuring the distance of the leading edgeof the dye from compared to the whole length of the small intestine 15minutes to two hours following oral dosing of the dye. Colonic motilityis assessed by measuring time to extrusion of a single glass beadinserted a standardized distance into the distal colon. The effect ofexample compounds on GIT motility in PD mice is calculated as theability to restore transit distance to those observed in control micefrom those observed in PD mice treated with vehicle, expressed as apercentage.

Example 262 Effect on Blood Pressure in a Models of Salt-SensitiveHypertension

Arterial blood pressure is measured to assess the ability of selectedexample compounds to attenuate hypertension in a model of salt-sensitivehypertension. Dahl Salt Sensitive (DSS) rats are a well characterizedmodel of salt-sensitive hypertension and end-organ injury.Salt-sensitive hypertension is established in DSS rats by increasing theNaCl content of the diet from 0.49% up to 4% NaCl for a period of 1 to4-weeks. DSS rats maintained on 0.49% NaCl are used as a control group.Animals are 6-10 weeks of age at study initiation, and have ad libitumaccess to food and water for the duration of the study and aremaintained in a temperature and humidity controlled room on a 12-hrlight/dark cycle. Rats (n=6-8/group) are dosed by oral gavage with testcompound (0.01-30 mg/kg) or vehicle (3 mM HCl, 0.01% Tween80) at a dosevolume of 5 mL/kg, twice daily for 1 to 3 weeks, while maintained on a4% NaCl diet. Arterial blood pressure is measured weekly by tail cuffplethysmography. A 24-hr urine collection is also collected weekly byplacing animals individually in metabolic cages.

Example 263 Effect on Cardiac Function in Models of Heart Failure

Serial echocardiography is used to measure cardiac function andmorphology to assess the ability of selected example compounds toimprove cardiac function, structure and neuro-humoral activation in arat model of heart failure. Male Dahl Salt Sensitive (DSS) rats or maleLewis rats are used to induce heart failure by permanent left maincoronary arterial ligation. Animals are 6-10 weeks of age at studyinitiation, and have ad libitum access to food and water for theduration of the study and are maintained in a temperature and humiditycontrolled room on a 12-hr light/dark cycle. Rats (n=6-10/group) aredosed by oral gavage with test compound (0.01-30 mg/kg) or vehicle (3 mMHCl, 0.01% Tween80) at a dose volume of 5 mL/kg, twice daily for 1 to 8weeks. Serial echocardiography is performed weekly to assesstime-dependent cardiac remodeling (HWI, LVI, chamber size),time-dependent cardiac performance (EF, dP/dt, LVEDP) changes andtime-dependent cardiac morphometry (HWI, LVI, LVEDV, LVESV) indices.Terminal assessment of load-dependent and load-independent leftventricular function are made using pressure-volume loop analysis.Extracellular volume expansion is assessed by measuring volume sensitivehormones ANP and BNP.

Example 264 Pain Relief in IBS-C—Reduction of Visceral Hypersensitivityin Rats

The ability of selected example compounds to reduce the hypersensitivityof the colon to balloon distension (CRD) in a rat model of visceralhypersensitive is measured by grading the rat's abdominal withdrawalreflex (AWR) and by measuring electromyographic (EMG) responses.Visceral hypersensitivity is induced by injecting 10-day old maleSprague Dawley rat pups with a 0.2 mL infusion of 0.5% acetic acidsolution in saline into the colon 2 cm from the anus. Control ratsreceive an equal volume of saline. Visceral hypersensitivity is thenassessed in these rats as adults, between 8 and 12 weeks of age. Rats(n=4-10/group) are dosed by oral gavage with test compound (0.01 to 30mg/kg) or vehicle (3 mM HCl, 0.01% Tween80) at a dose volume of 5 mL/kg,twice daily for up to 2 weeks prior to the assessment of visceralhypersensitivity. Visceral hypersensitivity is measured by grading theresponse to CRD. Under mild sedation with 1% methohexital sodium, aflexible balloon attached to Tygon tubing is inserted 8 cm into thedescending colon and rectum via the anus and secured in place by tapingthe tube to the tail. Approximately 30 minutes later, CRD is performedby rapidly inflating the balloon to varying pressures (10 to 80 mmHg)measured by a sphygmomanometer connected to a pressure transducer for a20 second period followed by a 2-minute rest period. Behavioralresponses to CRD are measured by grading the AWR by blinded observer andassigning an AWR score as follows: 1, normal behavior without response;2, contraction of abdominal muscles; 3, lifting of abdominal wall; 4,body arching and lifting of pelvic structures. EMG responses aremeasured continuously in response to CRD via two electrodes implanted atleast one-week prior to in the external oblique muscle and calculated asthe area under the curve of the EMG in response to CRD.

Topological Polar Surface Area Data

Topological Polar Surface Area (tPSA) values for representativecompounds in the disclosure are shown in Table 14, below. The tPSAvalues were calculated using the method of Ertl et al., Journal ofMedicinal Chemistry, 43:3714-3717 (2000).

TABLE 14 tPSA Values of Compounds Topological polar Example # surfacearea (A²) Example 01  125 Example 02  125 Example 03  125 Example 04 125 Example 05  125 Example 06  125 Example 07  121 Example 08  154Example 09  132 Example 10  125 Example 11  125 Example 12  125 Example13  125 Example 14  125 Example 15  124 Example 16  177 Example 17  134Example 18  116 Example 19  116 Example 20  116 Example 21  238 Example22  116 Example 23  116 Example 24  177 Example 25  238 Example 26  116Example 27  134 Example 28  112 Example 29  229 Example 30  137 Example31  137 Example 32  137 Example 33  137 Example 34  119 Example 35  119Example 36  119 Example 37  119 Example 38  112 Example 39  112 Example40  119 Example 41  291 Example 42  291 Example 43  309 Example 44  318Example 45  199 Example 46  387 Example 47  404 Example 48  224 Example49  417 Example 50  297 Example 51  213 Example 52  213 Example 53  213Example 54  213 Example 55  213 Example 56  213 Example 57  241 Example58  184 Example 59  220 Example 60  147 Example 61  134 Example 62  134Example 63  215 Example 64  134 Example 65  123 Example 66  147 Example67  161 Example 68  117 Example 69  117 Example 70  134 Example 71  208Example 72  154 Example 73  134 Example 74  174 Example 75  178 Example76  125 Example 77  238 Example 78  121 Example 79  123 Example 80  136Example 81  242 Example 82  112 Example 83  191 Example 84  190 Example85  123 Example 86  228 Example 87  270 Example 88  270 Example 89  159Example 90  189 Example 91  147 Example 92  147 Example 93   74 Example94  157 Example 95  115 Example 96  115 Example 97  312 Example 98  312Example 99  235 Example 100 212 Example 101 202 Example 102 487 Example103 212 Example 104 500 Example 168 251 Example 169 214 Example 170 270Example 171  86 Example 172 270 Example 173 185 Example 174 243 Example175 211 Example 176 233 Example 177 211 Example 178 220 Example 179 219Example 180 229 Example 181 229 Example 182 229 Example 183 211 Example184 202 Example 185 214 Example 186 237 Example 187 238 Example 188 211Example 189 231 Example 190 211 Example 191 211 Example 192 273 Example193 231 Example 194 221 Example 195 220 Example 196 211 Example 197 229Example 198 238 Example 199 229 Example 200 211 Example 201 220 Example202 235 Example 203 235 Example 204 290 Example 205 251 Example 206 177Example 207 251 Example 208 253 Example 209 253 Example 210 500 Example211 227 Example 212 445 Example 213 347 Example 214 176 Example 215 344Example 216 229 Example 217 441 Example 218 251 Example 219 280 Example220 280 Example 221 192 Example 222 270 Example 223 270 Example 224 270Example 225 270 Example 226 270 Example 227 270 Example 228 229 Example229 270 Example 230 229 Example 231 211 Example 232 194 Example 233 229Example 234 211 Example 235 194 Example 236 235 Example 237 235 Example238 235 Example 239 235 Example 240 270 Example 241 270 Example 242 270Example 243 270 Example 244 253 Example 245 253 Example 246 229 Example247 158 Example 248 253 Example 249 253 Example 250 212 Example 251 253Example 252 253

Pharmacological Data 1. Pharmacological Test Example 1

Cell-Based Assay of NHE-3 Activity.

Rat NHE-3-mediated Na⁺-dependent H⁺ antiport was measured using amodification of the pH sensitive dye method originally reported by Tsien(Proc. Natl. Acad. Sci. USA. (1984) 81(23): 7436-7440). Opossum kidney(OK) cells were obtained from the ATCC and propagated per theirinstructions. The rat NHE-3 gene was introduced into OK cells viaelectroporation, seeded into 96 well plates and grown overnight. Mediumwas aspirated from the wells, cells were washed twice with NaCl-HEPESbuffer (100 mM NaCl, 50 mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl₂, 1mM MgCl₂, pH 7.4), then incubated for 30 min at room temperature withNH₄Cl-HEPES buffer (20 mM NH₄Cl, 80 mM NaCl, 50 mM HEPES, 5 mM KCl, 2 mMCaCl₂), 1 mM MgCl₂, pH 7.4) containing 5 uM BCECF-AM (Invitrogen). Cellswere washed twice with Ammonium free, Nat-free HEPES (100 mM choline, 50mM HEPES, 10 mM glucose, 5 mM KCl, 2 mM CaCl₂), 1 mM MgCl₂, pH 7.4) andincubated in the same buffer for 10 minutes at room temperature to lowerintracellular pH. NHE-3-mediated recovery of neutral intracellular pHwas initiated by addition of Na-HEPES buffer containing 5 uM ethylisopropyl amiloride (EIPA, a selective antagonist of NHE-1 activity thatdoes not inhibit NHE-3) and 0-30 uM test compound, and monitoring the pHsensitive changes in BCECF fluorescence (λ_(ex) 505 nm, λ_(em) 538 nm)normalized to the pH insensitive BCECF fluorescence (λ_(ex) 439 nm,λ_(em) 538 nm). Initial rates were were plotted as the average 3-6replicates, and pIC₅₀ values were estimated using GraphPad Prism. Theinhibitory data of many of the example compounds illustrated above areshown in Table 15, below.

TABLE 15 Inhibitory data of compounds against rat NHE-3 rat NHE-3Example # Average pIC50¹ Example 171 <5.0 Example 174 <5.0 Example 175<5.0 Example 223 <5.0 Example 231 <5.0 Example 232 <5.0 Example 233 <5.0Example 235 <5.0 Example 30  5 to 6 Example 31  5 to 6 Example 52  5 to6 Example 54  5 to 6 Example 63  5 to 6 Example 64  5 to 6 Example 176 5to 6 Example 196 5 to 6 Example 209 5 to 6 Example 219 5 to 6 Example234 5 to 6 Example 28  6 to 7 Example 29  6 to 7 Example 45  6 to 7Example 46  6 to 7 Example 60  6 to 7 Example 65  6 to 7 Example 66  6to 7 Example 67  6 to 7 Example 68  6 to 7 Example 69  6 to 7 Example97  6 to 7 Example 100 6 to 7 Example 102 6 to 7 Example 104 6 to 7Example 169 6 to 7 Example 170 6 to 7 Example 178 6 to 7 Example 207 6to 7 Example 210 6 to 7 Example 211 6 to 7 Example 213 6 to 7 Example217 6 to 7 Example 218 6 to 7 Example 225 6 to 7 Example 228 6 to 7Example 47  >7 Example 81  >7 Example 87  >7 Example 88  >7 Example98  >7 Example 103 >7 Example 172 >7 Example 177 >7 Example 191 >7Example 195 >7 Example 200 >7 Example 201 >7 Example 202 >7 Example203 >7 Example 204 >7 Example 205 >7 Example 206 >7 Example 208 >7Example 212 >7 Example 215 >7 Example 216 >7 Example 222 >7 Example224 >7 Example 229 >7 Example 230 >7 Example 236 >7 Example 237 >7Example 244 >7 Example 250 >7 Example 251 >7 ¹pIC50 is the negative logthe IC50 value (an IC50 value of 1 micromolar corresponds to a pIC50value of 6.0)

2. Pharmacological Test Example 2

Parallel Artificial Membrane Permeability Assay (PAMPA).

The model consists of a hydrophobic filter material coated with amixture of lecithin/phospholipids creating an artificial lipid membrane.BD Gentest PAMPA 96-well plates (cat #353015) are warmed for 1 hr atroom temperature. 1 mL of 20 uM control compounds (pooled mix of 10 mMatenolol, ranitidine, labetalol, and propranolol) in transport buffer(10 mM HEPES in HBSS pH 7.4) are prepared along with 1 mL of 20 uM testcompounds in transport buffer. The PAMPA plates are separated, and 0.3mL of compound are added in duplicate to apical side (bottom/donorplate=“AP”), and 2 mL buffer are placed in the basolateral chamber(top/receiver plate=“BL”). The BL plate is placed on the AP plate andincubated for 3 hrs in 37° C. incubator. At that time, samples areremoved from both plates, and analyzed for compound concentration usingLC/MS. A “P_(e)” (effective permeability) value is calculated using thefollowing formula.

P _(e)=(−ln[1−C _(A)(t)/C _(eq)])/[A*(1/V _(D)+1/V _(A))*t

where

C_(A)=concentration in acceptor well, C_(D)=concentration in donor well

V_(D)=donor well volume (mL), V_(A)=acceptor well volume (mL)

A=filter area=0.3 cm², t=transport time (seconds)

C_(eq)=equilibriumconcentration=[C_(D)(t)*V_(D)+C_(A)(t)*V_(A)]/(V_(D)+V_(A))

P_(e) is reported in units of cm/sec×10⁻⁶.

Results from PAMPA testing are shown in Table 16.

TABLE 16 Papp values as determined using the PAMPA assay Avg Papp, A →B, Example # cm/sec × 10⁻⁶ Example 01 0.53 Example 03 0.8 Example 07 0.5Example 08 0.2 Example 13 0.3 Example 14 0.4 Example 15 0.05 Example 16<0.02 Example 23 <0.04 Example 24 0.03 Example 26 <0.02 Example 27 <0.02Example 30 0.56 Example 31 0.61 Example 34 0.2 Example 35 0.17 Example36 0.2 Example 37 0.1 Example 38 0.1 Example 44 0.1 Example 47 <0.01Example 48 0.9 Example 51 0.2 Example 52 1.61 Example 53 1.6 Example 541.3 Example 56 0.5 Example 57 1.65 Example 58 0.2 Example 59 0.1 Example60 0.99 Example 61 0.1 Example 63 0.43 Example 68 0.35 Example 69 0.3Example 70 0.4 Example 71 0.45 Example 72 0.2 Example 73 0.27 Example 740.45 Example 75 0.4 Example 76 0.2

Increasing values of tPSA are typically associated with lowerpermeability. FIG. 1 illustrates the Relationship between tPSA andPermeability (Papp, as measured in the PAMPA assay) of Examplecompounds. Compounds with higher tPSA values trend toward lowerpermeability.

3. Pharmacological Test Example 3

Pharmacodynamic Model: Effect of Test Compounds on Fluid Content ofIntestinal Compartments.

Normal female Sprague Dawley rats, 7 weeks old, were acclimated for atleast 2 days. The animals were fed ad lib through the experiment. Groupsof 5 rats were orally gavaged with 1.5 mL of water containing a negativecontrol compound or test compounds, adjusted to a concentration thatresults in a dose of 10 mg/kg. Six hours after dosing, rats wereeuthanized with isofluorane. The cecum and colon were ligated and thenremoved. After a brief rinse in saline and pat-drying, the segments wereweighed. The segments were then opened, and the contents collected andweighed. The collected contents were then dried, and weighed again. The% water content was reported as 100×((Ww−Wd)/Ww) where Ww is the weightof the wet contents, and Wd is the weight of the contents after drying.The differences between groups are evaluated by one way ANOVA withBonferroni post tests. Examples are shown in FIGS. 2A and 2B (whereinrats were dosed orally with 10 mg/kg of compound (Example or Control),and then after 6 hours, cecum and colon contents were removed, weighedand dried, and the % water in the contents was determined: *, P<0.05 and***, P<0.01 compared to control in ANOVA analysis).

4. Pharmacological Test Example 4

Determination of Compound C_(max) and AUC

Sprague-Dawley rats were orally gavaged with test article (2.5 mg/kg)and serum was collected at 0.5, 1, 2 and 4 h. Serum samples were treatedwith acetonitrile, precipitated proteins removed by centrifugation andsupernatants analyzed by LC/MS/MS and compared against a standard curveto determine compound concentration. Table 17 illustrates data from thepharmacokinetic profiling of selected example compounds. All compoundswere orally dosed at the dosage shown, and pharmacokinetic parametersdetermined as described in the text.

TABLE 17 Pharmacokinetic Profiling of Selected Example Compounds ActualOral Dose Cmax AUC Example (mg/kg) (ng/mL) (ng × hr/mL) Example 01  2.121 53 Example 16  1.6 71 159 Example 31  1.3 11 56 Example 35  2.2 2.4 5Example 50  2.3 93 242 Example 52  4.6 14 9 Example 55  2.2 9 23 Example60  2.4 2 0 Example 63  2.4 0 0 Example 211 0.7 <2.3 <3.0 Example 2121.5 <2.7 <4.4 Example 213 9.5 <5.0 <5.0 Example 214 2.6 <5.0 <5.0Example 215 7.7 <2.0 <2.0 Example 216 1.9 <4.0 <8.3 Example 217 9.1<10.0 <10.0 Example 204 10.9 <2.0 <2.0 Example 218 9 <1.0 <1.0 Example169 11 <3.5 <4.0 Example 205 10.7 <2.0 <2.0 Example 225 27 <3.5 <5.3Example 226 31 <3.0 <5.0 Example 172 26 <2.0 <2.0 Example 228 23 <5.0<5.0 Example 230 17 <5.0 <5.0 Example 173 28 23 19 Example 174 27 <5.4<5.0 Example 208 12 <5.0 <5.0 Example 231 23 <2.5 <3.0 Example 232 17<2.0 <2.0 Example 233 19 <2.6 <6.8 Example 234 22 <2.0 <2.0 Example 23511 <5.0 <5.0 Example 175 28 8 6 Example 177 14 <3.2 <4.0 Example 178 18<2.0 <2.0 Example 179 27 <16.0 <35.0 Example 180 25 <10.0 <19.0 Example181 28 <2.0 <2.0 Example 185 17 <2.0 <2.0 Example 186 15 <3.4 <5.0Example 244 16 <7.0 <15.0 Example 245 21 <2.0 <2.0

5. Pharmacological Test Example 5

Evaluation of NHE-3-Inhibitory Compounds in Disease Models with Na/H₂ORetention: CRF/ESRD Model.

Male Sprague-Dawley rats with subtotal (⅚^(th)) nephrectomy, 7 weeks oldand weighing 175-200 g at surgery time, are purchased from Charles RiverLaboratories. The animals are subjected to acclimation for 7 days, andrandomly grouped (using random number table) before proceeding toexperiments. During acclimation, all animals are fed with base dietHD8728CM. The rats are housed in holding cages (2/cage) during theacclimation period and the time between sample collections. The rats aretransferred to metabolic cages on the days of sample collections. Foodand water is provided ad libitum.

Chronic renal failure is induced in the rats by subtotal (⅚th)nephrectomy (Nx) followed by intravenous (IV) injection of adriamycin(ADR) at 2 weeks post-nephrectomy, at a dose of 3.5 mg/kg body weight.Animals are then random/zed into control and treatment groups with 10rats per group. Rats in untreated group are fed with base diet and ratsin the treatment groups are fed the same chow supplemented with NHE-3inhibitor/fluid holding polymer at various doses. All the groups aremaintained for 28 days.

Serum samples are collected at day (−1) (1 days before ADR injection),days 14 and 28 post ADR treatment. Twenty four hour urine and fecalsamples are collected at day (−1), days 14 and 28 post ADR treatment andstored at −20° C. for later analysis. Body weight, food and waterconsumption are measured at the same time points as urine collections.Serum and urine chemistry (Na, K, Ca, Cl) are determined using an ACEClinical Chemistry System (ALFA WASSER MANN Diagnostic Technologies,LLC). Fecal electrolyte (Na, K, Ca, Cl) excretions are determined by IC.Fluid balance are also determined via amount of fluid intake (indrinking water) subtracted by combined fecal water amount and urinevolume. Tissues (heart, kidney and small intestine) are harvested at theend of experiments for later histopathological analysis. The third space(pleural fluids and ascites) body fluid accumulation are scoredsemi-quantitatively as follows: grade 0, no fluid accumulation; grade 1,trace amount of fluids; grade 2, obvious amount of fluids; grade 3, bothcavities full of fluids; grade 4, fluids overflowed once the cavitiesare opened. Each score of body fluid accumulation is confirmed andagreed on by 2 investigators.

Animals treated with NHE-3 inhibitor/fluid holding polymer showdecreased serum aldosterone, decreased 24 hr urine volume and decreasedurine K excretion, and increased urine Na excretion compared to notreatment group. Treated animals also have increased fecal Na and fluidexcretion, compared to control group. Compared to untreated rats whichshow positive fluid balance of 4 g per day, animals treated with NHE-3inhibitor/fluid holding polymer demonstrate a fluid loss of 5 g per day.

Treatment of NHE-3 inhibitor/fluid holding polymer in CRF rats isassociated with less edema in heart, kidney and small intestine tissues,less hypertrophy in heart, less third space fluid accumulation, andlower body weight at the end of experiment compared to untreated group.

6. Pharmacological Test Example 6

Evaluation of NHE-3-inhibitory Compounds in Disease Models with Na/H₂ORetention: Congestive Heart Failure Model.

CHFs are introduced to male Spraque Dawley rats, 7-8 weeks old fed adlib regular diet and ad lib 10% ethanol in drinking water, and gavagedwith a daily dose of 6.3 mg cobalt acetate for 7 days. Then CHF rats aregavaged with a daily dose of 4 mg of furosemide for 5 days, inducingresistance to furosemide diuretic effects. The rats are then randomlydivided into 2 groups, control and treatment, and the treatment groupadministered NHE-3 inhibitor/fluid holding polymer for 7 days. Day 0 andday 7 post treatment serum aldosterone levels, urine volume, urine Naand K excretions are measured. Fluid balance is also determined viaamount of fluid intake (in drinking water) subtracted by combined fecalfluid amount and urine volume.

Animals treated with NHE-3 inhibitor/fluid holding polymer havedecreased serum aldosterone levels, decreased 24 hr urine volume andurine K excretion, and increased urine Na excretion compared to controlgroup. Animals treated with NHE-3 inhibitor/fluid holding polymer have,for example, increased fecal Na and fluid excretion. Compared tountreated rats, which show a positive fluid balance of, for example, 4 gper day, treated animals demonstrate a fluid loss of 5 g per day.

7. Pharmacological Test Example 7

Evaluation of NHE-3-inhibitory Compounds in Disease Models with Na/H₂ORetention: Hypertension Model. Male Dahl salt-sensitive rats areobtained from Harlan Teklad. After acclimation, animals are randomlygrouped and fed diet containing 8% NaCl±NHE-3 inhibitor/fluid holdingpolymer for 7 days. Day 0 and day 7 post treatment systolic BP, serumaldosterone levels, urine volume, urine Na and K excretions aremeasured. Fluid balance is also determined via amount of fluid intake(in drinking water) subtracted by combined fecal fluid amount and urinevolume.

Animals treated with NHE-3 inhibitor/fluid holding polymer would showdecreased systolic BP, serum aldosterone levels, 24 hr urine volume andurine K excretion, and increased urine Na excretion compared to notreatment group. Animals treated with NHE-3 inhibitor/fluid holdingpolymer would also show increased fecal fluid excretion. Compared tountreated rats which would show positive fluid balance of 4 g per day,animals treated with NHE-3 inhibitor/fluid holding polymer demonstrate afluid loss of 2 g per day.

8. Pharmacological Test Example 8

Na Transport Inhibition Study on Colonic Tissues. Immediately followingeuthanasia and exsanguinations of the rats, the entire distal colon isremoved, cleansed in ice-cold isotonic saline, and partially stripped ofthe serosal muscularis using blunt dissection. Flat sheets of tissue aremounted in modified Ussing chambers with an exposed tissue area of 0.64cm². Transepithelial fluxes of ²²Na⁺ (Perkin Elmer Life Sciences,Boston, Mass.) are measured across colonic tissues bathed on both sidesby 10 ml of buffered saline (pH 7.4) at 37° C. and circulated bybubbling with 95% O₂-5% CO₂. The standard saline contains the followingsolutes (in mmol/l): 139.4 Na⁺, 5.4 K, 1.2 Mg²⁺, 123.2 Cl⁻, 21.0 HCO₃ ⁻,1.2 Ca²⁺, 0.6 H₂PO₄ ⁻, 2.4 HPO²⁻, and 10 glucose. The magnitude anddirection of the net flux (Jnet Na) is calculated as the differencebetween the two unidirectional fluxes (mucosal to serosal, Jms Na andserosal to mucosal, Jsm Na) measured at 15-min intervals for a controlperiod of 45 min (Per I), under short-circuit conditions. In someseries, Per I is followed by a second 45-min flux period (Per II) todetermine the acute effects of NHE inhibitors.

9. Pharmacological Test Example 9

Pharmacodynamic Model: Effect of Test Compounds and FAP on Consistencyand Form of Rat Stools.

Normal rats are given a NHE-3 inhibiting compound and optionally afluid-absorbing or -holding polymer mixed in their diet at escalateddoses. Distilled water is available at libitum. Clinical data monitoredare body weight, food intake, water intake, fecal and urinary output.Urinary Na, K and creatinine are measured by a Clinical Analyzer(VetAce; Alfa Wassermann Diagnostic Technologies. LLC, West Caldwell,N.J.). The consistency of the stools expelled within 24 h after theadministration of each drug or vehicle is reported as follows: when thefeces are unformed, i.e., muddy or watery, this is judged to be diarrheaand the percentage diarrhea is reported as the ratio of the number ofanimals producing unformed stools to the number tested. All of the fecesis collected just after each evacuation and put into a covered vesselprepared for each animal in order to prevent the feces from drying. Toinvestigate the duration of activity of each drug, the feces collectedover each 8-h period is dried for more than 8 h at 70° C. in aventilated oven after the wet weight is measured. The fecal fluidcontent is calculated from the difference between the fecal wet weightand the dry weight. Fecal Na and K is analyzed by ion Chromatography(Dionex) after acid digestion of the feces specimen.

10. Pharmacological Test Example 10

Effect of Test Compounds and FAP on CKD Rats.

Male Sprague-Dawley rats (275-300 g; Harlan, Indianapolis, Ind.) areused and have free access to water and Purina rat chow 5001 at alltimes. A ⅚ nephrectomy is performed to produce a surgical resection CRFmodel and the treatment study is performed 6 wk after this procedure. Inone control group, CRF rats are given access to Purina rat chow; intreated groups. CRF rats are given access to Purina rat chow mixed withthe article, i.e. a NHE-3 inhibiting compound and optionally afluid-absorbing or -holding polymer. The treatment period is 30 days.Systolic blood pressure is monitored in all animals with the use of atail sphygmomanometer (Harvard Apparatus, South Natick, Mass.). All ratsare euthanatized by an intraperitoneal injection of pentobarbital (150mg/kg body wt), and blood is collected by cardiac puncture for serum Na⁺(Roche Hitachi Modular P800 chemistry analyzer; Roche Diagnostics,Indianapolis, Ind.) and creatinine determination (kit 555A; SigmaChemical. St. Louis, Mo.). Sodium and creatinine is also determined in aurine specimen collected over 24 h immediately before euthanasia.

11. Pharmacological Test Example 11

Effect of Test Compounds on Intestinal Fluid Accumulation in SucklingMice.

Institute of Cancer Research/Harlan Sprague-Dawley (ICR-HSD) sucklingmice, 2 to 4 days old (2.1±1.0 g), are dosed orally with 0.1 mL of testsolution (vehicle (1 mmol/L HEPES) or NHE inhibitor dissolved invehicle). After dosing, the mice are kept at room temperature for 3hours, then killed, the intestinal and body weights measured, and aratio of the intestinal weight to remaining body weight is calculated. Aratio of 0.0875 represents one mouse unit of activity, indicatingsignificant fluid accumulation in the intestine.

12. Pharmacological Test Example 12

Determination of Water-absorbing Capacity.

This test is designed to measure the ability of a polymer to absorb 0.9%saline solution against a pressure of 50 g/cm² or 5 kPa. Thesuperabsorbent is put into a plastic cylinder that has a screen fabricas bottom. A weight giving the desired pressure is put on top. Thecylinder arrangement is then placed on a liquid source. Thesuperabsorbent soaks for one hour, and the absorption capacity isdetermined in g/g.

This test principle is described in the European Disposables AndNonwovens Association (EDANA) standard EDANA ERT 442 GravimetricDetermination of Absorption under Pressure or Absorbency Under Load(AUL), or in the AUL-test found in column 12 in U.S. Pat. No. 5,601,542,the entire contents of which are incorporated herein by reference forall relevant and consistent purposes. Any of these two methods can beused, or the simplified method described below.

Equipment:

-   -   A plastic cylinder having a screen fabric made of steel or nylon        glued to the bottom. The fabric can have mesh openings of 36 μm        (designated “400 mesh”), or in any case smaller than the        smallest tested particles. The cylinder can have an internal        diameter of 25.4 mm, and a height of 40 mm. A larger cylinder        can also be used, such as the apparatus in the EDANA standard        ERT 442—Gravimetric Determination of Absorption under Pressure.    -   A plastic piston or spacer disc with a diameter slightly smaller        than the cylinder's inner diameter. For a cup with a 25.4 mm        inner diameter the disc can be 25.2 mm wide, 8 mm high, and        weigh about 4.4 g.    -   A weight that exerts a 50 g/cm² pressure on the superabsorbent        (in combination with the piston). For a 25.4 mm inner diameter        cylinder (=5.067 cm²) and a 4.4 g piston, the weight should have        a mass of 249 g.    -   Glass or ceramic filter plate (porosity=0). The plate is at        least 5 mm high, and it has a larger diameter than the cylinder.    -   Filter paper with a larger diameter than the cylinder. Pore size        <25 μm.    -   Petri dish or tray    -   0.9% NaCl solution

Procedure:

-   -   Put the glass filter plate in a Petri dish, and place a filter        paper on top.    -   Fill the Petri dish with 0.9% NaCl solution—up to the edge of        the filter plate.    -   Weigh a superabsorbent sample that corresponds to a 0.032 g/cm²        coverage on the cylinder's screen fabric (=0.16 g for a cylinder        with a 25.4 mm inner diameter). Record the exact weight of the        sample (A). Carefully distribute the sample on the screen        fabric.    -   Place the plastic piston on top of the distributed sample, and        weigh the cylinder assembly (B). Then mount the weight onto the        piston.    -   Place the assembly on the filter paper, and let the        superabsorbent soak for 60 minutes.    -   Remove the weight, and weigh the assembly with the swollen        superabsorbent (C).    -   Calculate the AUL in g/g according to this formula: C-B.

13. Pharmacological Test Example 13

Pharmacodynamic Model: Effect of Test Compounds on Fecal Water Content.

Normal female Sprague Dawlev rats (Charles-River laboratoriesinternational, Hollister, Calif.), 7-8 weeks old with body weight175-200 g were acclimated for at least 3 days before proceeding toexperiments. The animals were provided food (Harlan Teklad 2018c) andwater ad lib, through the experiment. Animals were randomly grouped with6 rats per group.

The experiments were initiated by orally dosing test compounds at 3mg/kg in volume of 10 ml/kg. Rats from control group were gavaged withthe same volume of vehicle (water). After dosing, rats were placed inmetabolic cages for 16 hrs (overnight). Food and water consumption weremonitored. After sixteen hours, feces and urine were collected. Thepercent of fecal water was measured by weighing fecal samples before andafter drying.

Representative data of % fecal water content are shown in Table 12 (dataare expressed as means, with 6 animals per data point). The differencesbetween control and treated groups were evaluated by one way ANOVA withDunnett post tests. Results are significant if p<0.05.

TABLE 12 % Fecal % Fecal water (% of Example water control) Significant?224 65% 125% Y 234 58% 117% Y 239 58% 114% Y 178 59% 118% Y 237 60% 120%Y 238 60% 121% Y 177 60% 121% Y 244 61% 118% Y 236 64% 128% Y 250 60%120% Y 200 62% 124% Y 201 63% 127% Y 202 63% 134% Y 203 61% 130% Y

14. Pharmacological Test Example 14

Pharmacodynamic Model: Effect of Test Compounds on Urinary SodiumLevels.

It is anticipated that the reduction of absorption of sodium from theintestine will be reflected in reduced levels of sodium in the urine. Totest this, the protocols in Example 13 were repeated, but urine wascollected in addition to feces. Urine sodium levels were analyzed by ionchromatography (IC), and the amound of sodium excreted in the urine wascorrected for variations in sodium intake by measuring food consumption.In addition, test compounds were administered at several dose levels todemonstrate a dose-response relationship. As shown in FIGS. 3A and 3Bfor Examples 201, 244, and 260, where as rats excrete about half thesodium they consume in urine, in rats treated with increasing doses ofNHE-3 inhibitor, the amount of sodium excreted in the urine diminishessignificantly and dose dependently.

15. Pharmacological Test Example 15

Pharmacodynamic Model: Dose Dependent Effect of Test Compound on FecalWater Content.

Rats were monitored for fecal water content as in Example 13, and thetest compound was administered at several dose levels to demonstrate adose-response relationship. As shown in FIG. 4, in rats treated withincreasing doses of the NHE-3 inhibitor tested (i.e., Example 87), thefecal water content increased significantly and dose dependently.

16. Pharmacological Test Example 16

Pharmacodynamic Model: Addition of a Fluid Absorbing Polymer to Chow.

Rats were monitored for fecal water content as in Example 13, with theaddition of a second group that were fed chow with the addition of 1%Psyllium to their diet. In addition to fecal water and urinary sodium,fecal form was monitored on a scale of 1-5, where 1 is a normal pellet,3 indicates soft and unformed pellets, and 5 indicates watery feces. Asshown in FIGS. 5A, 5B and 5C, supplementing the diet with Psylliumresulted in a slight reduction of fecal stool form, but withoutimpacting the ability of the test compound (i.e., Example 224) toincrease fecal water content or decrease urinary sodium.

17. Pharmacological Test Example 17

Pharmacodynamic Model: Effect of Test Compounds on Acute Stress-InducedVisceral Hypersensitivity in Female Wistar Rats.

Female Wistar rats weighing 220-250 g were prepared forelectromyography. The animals were anaesthetized, and three pairs ofnichrome wire electrodes were implanted bilaterally in the striatedmuscles at 3 cm laterally from the midline. The free ends of electrodeswere exteriorised on the back of the neck and protected by a glass tubeattached to the skin. Electromyographic recordings (EMG) were begun 5days after surgery. The electrical activity of the abdominal striatedmuscles were recorded with an electromyograph machine (Mini VIII; Alvar,Paris, France) using a short time constant (0.03 sec.) to removelow-frequency signals (<3 Hz).

Partial restraint stress (PRS), a relatively mild stress, was performedas follows. Briefly, animals were lightly anaesthetized withethyl-ether, and their freeholders, upper forelimbs and thoracic trunkwere wrapped in a confining harness of paper tape to restrict, but notprevent their body movements and placed in their home cage for 2 hours.Control sham-stress animals were anaesthetized but not wrapped. PRS wasperformed between 10:00 and 12:00 AM.

Colorectal distension (CRD) was accomplished as follows: rats wereplaced in a plastic tunnel, where they were not allowed to move orescape daily during 3 consecutive days (3 h/day) before any CRD. Theballoon used for distension was 4 cm in long and made from a latexcondom inserted in the rectum at 1 cm of the anus and fixed at the tail.The balloon, connected to a barostat was inflated progressively by stepsof 15 mmHg, from 0, 15, 45 and 60 mmHg, each step of inflation lasting 5min. CRD was performed at T+2 h15 as a measure of PRS induced visceralhyperalgesia±test compound or vehicle. To determine the antinociceptiveeffect of test compounds on stress-induced visceral hypersensitivity,test compounds were administered 1 h before CRD in 6 groups of 8 femalerats. For each parameter studied (the number of abdominal contractionsfor each 5-min period during rectal distension) data is expressed asmean±SEM. Comparisons between the different treatments were performedusing an analysis of variance (ANOVA) followed by a Dunnett post test.The criterion for statistical significance is p<0.05.

FIG. 6 shows the results of this test using the compound illustrated inExample 224 dosed orally at 10 mg/kg, and shows that at 45 and 60 mm Hg,inhibition of NHE-3 in rats surprisingly reduces visceralhypersensitivity to distension (p<0.05).

18. Pharmacological Test Example 18

Pharmacodynamic Model: Effect of Test Compounds on Fecal Sodium Levels.

It is anticipated that the reduction of absorption of sodium from theintestine will be reflected in increase levels of sodium in the feces.To test this, the protocols in Example 13 were repeated. After drying offeces to determine water content, 1M HCl was added to dried ground fecesto a concentration of 50 mg/mL and extracted at room temperature onrotator for 5 days. Sodium content was analyzed by ion chromatography(IC). As shown in FIGS. 7A and 7B for Example 224, in rats treated withan NHE-3 inhibitor, the amount of sodium excreted in the fecessignificantly (p<0.05 by t-test).

19. Pharmacological Test Example 19

Determination of Compound Remaining in Feces.

Sprague-Dawley rats were orally gavaged with test article. A low dose ofcompound (0.1 mg/kg) was selected so that feces would remain solid andpractical to collect. For both Examples 202 and 203, three rats weredosed, and following dosage of compounds, the rats were placed inmetabolic cages for 72 hours. After 72 hours, fecal samples wererecovered and dried for 48 hours. Dried fecal samples were ground to apowdered from, and for each rat, 10 replicates of 50 mg samples wereextracted with acetonitrile. Insoluble materials were removed bycentrifugation and supernatants analyzed by LC/MS/MS and comparedagainst a standard curve to determine compound concentration. The amountof compound actually dosed was determined by LC/MS/MS analysis of thedosing solutions. The total amount of compound present in the 72-hourfecal samples was compared to the total amount of compound dosed, andreported as percentage of total dose recovered. The results, shown inTable 13, demonstrate near quantitative recovery of Examples 202 and 203in 72-hour fecal samples.

TABLE 13 Recovery of dosed compounds from 72-hour fecal samples %Recovery ± SD Example 202 Example 203 Rat 1 93.8 ± 11.8 100.3 ± 6.7  Rat2 90.5 ± 5.5  75.8 ± 8.2  Rat 3 92.4 ± 10.6 104.4 ± 7.1 

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification areincorporated herein by reference, in their entirety to the extent notinconsistent with the present description.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. A compound having: (i) a tPSA of at least about200 Å² and a molecular weight of at least about 710 Daltons in thenon-salt form; or (ii) a tPSA of at least about 270 Å², wherein thecompound is substantially active in the gastrointestinal tract toinhibit NHE-mediated antiport of sodium ions and hydrogen ions thereinupon administration to a patient in need thereof.
 2. A compound of claim1 wherein the compound has a molecular weight of at least about 500 Da.3. A compound of claim 1 or 2 wherein the compound has a molecularweight of at least about 1000 Da.
 4. A compound of any of claims 1-3wherein the compound has a molecular weight of at least about 2500 Da.5. A compound of any of claims 1-4 wherein the compound has a molecularweight of at least about about 5000 Da.
 6. A compound of any of claims1-5 wherein the compound has a tPSA of at least about 250 Å².
 7. Acompound of any of claims 1-6 wherein the compound has a tPSA of atleast about 270 Å².
 8. A compound of any of claims 1-7 wherein thecompound has a tPSA of at least about 300 Å².
 9. A compound of any ofclaims 1-8 wherein the compound has a tPSA of at least about 350 Å². 10.A compound of any of claims 1-9 wherein the compound has a tPSA of atleast about 400 Å².
 11. A compound of any of claims 1-10 wherein thecompound has a tPSA of at least about 500 Å².
 12. A compound of any ofclaims 1-11 wherein the compound is substantially active on the apicalside of the epithelium of the gastrointestinal tract to inhibit antiportof sodium ions and hydrogen ions mediated by NHE-3, NHE-2, NHE-8, or acombination thereof.
 13. A compound of any of claims 1-12 wherein thecompound is substantially systemically non-bioavailable and/orsubstantially impermeable to the epithelium of the gastrointestinaltract.
 14. A compound of any of claims 1-13 wherein the compound issubstantially active in the lower gastrointestinal tract.
 15. A compoundof any of claims 1-14 wherein the compound has (i) a total number of NHand/or OH and/or other potential hydrogen bond donor moieties greaterthan about 5: (ii) a total number of O atoms and/or N atoms and/or otherpotential hydrogen bond acceptors greater than about 10; and/or (iii) aMoriguchi partition coefficient greater than about 10⁵ or less thanabout
 10. 16. A compound of any of claims 1-15 wherein the compound hasa permeability coefficient, P_(app) of less than about 100×10⁻⁶ cm/s, orless than about 10×10⁻⁶ cm/s, or less than about 1×10⁻⁶ cm/s, or lessthan about 0.1×10⁻⁶ cm/s.
 17. A compound of any of claims 1-16 whereinthe compound is substantially localized in the gastrointestinal tract orlumen.
 18. A compound of any of claims 1-17 wherein the compoundinhibits NHE irreversibly.
 19. A compound of any of claims 1-18 whereinthe compound is capable of providing a substantially persistentinhibitory action and wherein the compound is orally administeredonce-a-day.
 20. A compound of any of claims 1-19 wherein the compound issubstantially stable under physiological conditions in thegastrointestinal tract.
 21. A compound of any of claims 1-20 wherein thecompound is inert with regard to gastrointestinal flora.
 22. A compoundof any of claims 1-21 wherein the compound is designed to be deliveredto the lower part of the gastrointestinal tract.
 23. A compound of anyof claims 1-22 wherein the compound is designed to be delivered to thelower part of the gastrointestinal tract past the duodenum.
 24. Acompound of any of claims 1-23 wherein the compound, when administeredat a dose resulting in at least a 10% increase in fecal water content,has a C_(max) that is less than the IC₅₀ for NHE-3, less than about 10×the IC₅₀, or less than about 100× the IC₅₀.
 25. A compound of any ofclaims 1-24 wherein, upon administration of the compound to a patient inneed thereof, the compound exhibits a maximum concentration detected inthe serum, defined as C_(max), that is lower than the NHE inhibitoryconcentration IC₅₀ of the compound.
 26. A compound of any of claims 1-24wherein, upon administration of the compound to a patient in needthereof, greater than about 80%, greater than about 90% or greater thanabout 95% of the amount of compound administered is present in thepatient's feces.
 27. A compound of any of claims 1-26 wherein thecompound has a structure of Formula (I) or (IX):

wherein: NHE is a NHE-inhibiting small molecule that comprises (i) ahetero-atom containing moiety, and (ii) a cyclic or heterocyclicscaffold or support moiety bound directly or indirectly thereto, theheteroatom-containing moiety being selected from a substitutedguanidinyl moiety and a substituted heterocyclic moiety, which mayoptionally be fused with the scaffold or support moiety to form a fusedbicyclic structure; and, Z is a moiety having at least one site thereonfor attachment to the NHE-inhibiting small molecule, the resulting NHE-Zmolecule possessing overall physicochemical properties that render itsubstantially impermeable or substantially systemicallynon-bioavailable; and, E is an integer having a value of 1 or more. 28.A compound of claim 27 wherein the total number of freely rotatablebonds in the NHE-Z molecule is at least about
 10. 29. A compound ofclaim 27 or 28 wherein the total number hydrogen bond donors in theNHE-Z molecule is at least about
 5. 30. A compound of any of claims27-29 wherein the total number of hydrogen bond acceptors in the NHE-Zmolecule is at least about
 10. 31. A compound of any of claims 27-30wherein the total number of hydrogen bond donors and hydrogen bondacceptors in the NHE-Z molecule is at least about
 10. 32. A compound ofany of claims 27-31 wherein the Log P of the NHE-Z inhibiting compoundis at least about
 5. 33. A compound of any of claims 27-31 wherein thelog P of the NHE-Z inhibiting compound is less than about 1, or lessthan about
 0. 34. A compound of any of claims 27-33 wherein the scaffoldis a 5-member or 6-member cyclic or heterocyclic moiety.
 35. A compoundof claim 34 wherein the scaffold is aromatic.
 36. A compound as setforth in any one of claims 27-35, wherein the scaffold of theNHE-inhibiting small molecule is bound to the moiety, Z, the compoundhaving the structure of Formula (II):

wherein: Z is a Core having one or more sites thereon for attachment toone or more NHE-inhibiting small molecules, the resulting NHE-Z moleculepossessing overall physicochemical properties that render itsubstantially impermeable or substantially systemicallynon-bioavailable; B is the heteroatom-containing moiety of theNHE-inhibiting small molecule, and is selected from a substitutedguanidinyl moiety and a substituted heterocyclic moiety, which mayoptionally be fused with the Scaffold moiety to form a fused, bicyclicstructure; Scaffold is the cyclic or heterocyclic scaffold or supportmoiety of the NHE-inhibiting small molecule, which is bound directly orindirectly to heteroatom-containing moiety, B, and which is optionallysubstituted with one or more additionally hydrocarbyl orheterohydrocarbyl moieties; X is a bond or a spacer moiety selected froma group consisting of substituted or unsubstituted hydrocarbyl orheterohydrocarbyl moieties, and in particular substituted orunsubstituted C₁₋₇ hydrocarbyl or heterohydrocarbyl, and substituted orunsubstituted, saturated or unsaturated, cyclic or heterocyclicmoieties, which links B and the Scaffold; and, D and E are integers,each independently having a value of 1 or more.
 37. A compound of any ofclaims 27-36 wherein the compound is an oligomer, dendrimer or polymer,and further wherein Z is a Core moiety having two or more sites thereonfor attachment to multiple NHE-inhibiting small molecules, eitherdirectly or indirectly through a linking moiety, L, the compound havingthe structure of Formula (X):CoreL-NHE)_(n)   (X) wherein L is a bond or linker connecting the Coreto the NHE-inhibiting small molecule, and n is an integer of 2 or more,and further wherein each NHE-inhibiting small molecule may be the sameor differ from the others.
 38. A compound of claim 37 wherein theNHE-inhibiting small molecule has the structure of Formula (IV):

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein: each R₁, R₂, R₃, R₅ and R₉ are independently selected from H,halogen, —NR₇(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇,—SR₇, —O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or a bond linking the NHE-inhibiting smallmolecule to L, provided at least one is a bond linking theNHE-inhibiting small molecule to L; R₄ is selected from H, C₁-C₇ alkyl,or a bond linking the NHE-inhibiting small molecule to L; R₆ is absentor selected from H and C₁-C₇ alkyl; and Ar1 and Ar2 independentlyrepresent an aromatic ring or a heteroaromatic ring.
 39. A compound ofclaim 38 wherein the NHE-inhibiting small molecule has the followingstructure:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof,wherein: each R₁, R₂ and R₃ are independently selected from H, halogen,—NR₇(CO)R₈, —(CO)NR₇R₈, —SO₂—NR₇R₈, —NR₇SO₂R₈, —NR₇R₈, —OR₇, —SR₇,—O(CO)NR₇R₈, —NR₇(CO)OR₈, and —NR₇SO₂NR₈, where R₇ and R₈ areindependently selected from H or a bond linking the NHE-inhibiting smallmolecule to L, provided at least one is a bond linking theNHE-inhibiting small molecule to L.
 40. A compound of claim 39 whereinthe NHE-inhibiting small molecule has one of the following structures:

or a stereoisomer, prodrug or pharmaceutically acceptable salt thereof.41. A compound of any of claims 37-40 wherein L is a polyalkylene glycollinker.
 42. A compound of any of claims 37-41 wherein L is apolyethylene glycol linker.
 43. A compound of any of claims 37-42wherein n is
 2. 44. A compound of any of claims 37-43 wherein the Corehas the following structure:

wherein: X is selected from the group consisting of a bond, —O—, —NH—,—S—, C₁₋₆alkylene, —NHC(═O)—, —C(═O)NH—, —NHC(═O)NH—, —SO₂NH—, and—NHSO₂—; Y is selected from the group consisting of a bond, optionallysubstituted C₁₋₈alkylene, optionally substituted aryl, optionallysubstituted heteroaryl, a polyethylene glycol linker,—(CH₂)₁₋₆O(CH₂)₁₋₆— and —(CH₂)₁₋₆NY₁(CH₂)₁₋₆—; and Y₁ is selected fromthe group consisting of hydrogen, optionally substituted C₁₋₈alkyl,optionally substituted aryl or optionally substituted heteroaryl.
 45. Acompound of any of claims 37-44 wherein the Core is selected from thegroup consisting of:


46. A compound of any of claims 27-36 wherein the compound is anoligomer, and further wherein Z is a linking moiety, L, that links twoor more NHE-inhibiting small molecules together, when the two or moreNHE-inhibiting small molecules may be the same or different, thecompound having the structure of Formula (XI):

wherein L is a bond or linker connecting one NHE-inhibiting smallmolecule to another, and m is 0 or an integer of 1 or more.
 47. Acompound of any of claims 27-36 wherein the compound is an oligomer,dendrimer or polymer, and further wherein Z is a backbone, denotedRepeat Unit, to which is bound multiple NHE-inhibiting moieties, thecompound having the structure of Formula (XIIB):

wherein: L is a bond or a linking moiety; NHE is a NHE-inhibiting smallmolecule; and n is a non-zero integer.
 48. A pharmaceutical compositioncomprising a compound of any of claims 1-47, or a stereoisomer,pharmaceutically acceptable salt or prodrug thereof, and apharmaceutically acceptable carrier, diluent or excipient.
 49. Apharmaceutical composition of claim 48, further comprising afluid-absorbing polymer.
 50. A pharmaceutical composition of claim 49wherein the fluid-absorbing polymer is delivered directly to the colon.51. A pharmaceutical composition of claim 49 or 50 wherein thefluid-absorbing polymer has a fluid absorbency of at least about 15 g ofisotonic fluid per g of polymer under a static pressure of about 5 kPa.52. A pharmaceutical composition of any of claims 49-51 wherein thefluid-absorbing polymer has a fluid absorbency of at least about 15 g ofisotonic fluid per g of polymer under a static pressure of about 10 kPa.53. A pharmaceutical composition of any of claims 49-52 wherein thefluid-absorbing polymer is characterized by a fluid absorbency of atleast about 10 g/g.
 54. A pharmaceutical composition of any of claims49-53 wherein the fluid-absorbing polymer is characterized by a fluidabsorbency of at least about 15 g/g.
 55. A pharmaceutical composition ofany of claims 49-54 wherein the fluid-absorbing polymer issuperabsorbent.
 56. A pharmaceutical composition of any of claims 49-54wherein the fluid-absorbing polymer is a crosslinked, partiallyneutralized polyelectrolyte hydrogel.
 57. A pharmaceutical compositionof any of claims 49-54 wherein the fluid-absorbing polymer is acrosslinked polyacrylate.
 58. A pharmaceutical composition of any ofclaims 49-54 wherein the fluid-absorbing polymer is a polyelectrolyte.59. A pharmaceutical composition of any of claims 49-54 wherein thefluid-absorbing polymer is calcium Carbophil.
 60. A pharmaceuticalcomposition of any of claims 49-54 wherein the fluid-absorbing polymeris prepared by a high internal phase emulsion process.
 61. Apharmaceutical composition of any of claims 49-54 wherein thefluid-absorbing polymer is a foam.
 62. A pharmaceutical composition ofany of claims 49-54 wherein the fluid-absorbing polymer is prepared by aaqueous free radical polymerization of acrylamide or a derivativethereof, a crosslinker and a free radical initiator redox system inwater.
 63. A pharmaceutical composition of any of claims 49-54 whereinthe fluid-absorbing polymer is a hydrogel.
 64. A pharmaceuticalcomposition of any of claims 49-54 wherein the fluid-absorbing polymeris an N-alkyl acrylamide.
 65. A pharmaceutical composition of any ofclaims 49-54 wherein the fluid-absorbing polymer is a superporous gel.66. A pharmaceutical composition of any of claims 49-54 wherein thefluid-absorbing polymer is naturally occurring.
 67. A pharmaceuticalcomposition of any of claims 49-54 wherein the fluid-absorbing polymeris selected from the group consisting of xanthan, guar, wellan,hemicelluloses, alkyl-cellulose hydro-alkyl-cellulose,carboxy-alkyl-cellulose, carrageenan, dextran, hyaluronic acid andagarose.
 68. A pharmaceutical composition of any of claims 49-54 whereinthe fluid-absorbing polymer is psyllium.
 69. A pharmaceuticalcomposition of any of claims 49-54 wherein the fluid-absorbing polymeris a polysaccharide that includes xylose and arabinose.
 70. Apharmaceutical composition of any of claims 49-54 wherein thefluid-absorbing polymer is a polysaccharide that includes xylose andarabinose, wherein the ratio of xylose to arabinose is at least about3:1, by weight.
 71. A pharmaceutical composition of any of claims 49-70,further comprising another pharmaceutically active agent or compound.72. A pharmaceutical composition of claim 71 wherein the compositionfurther comprises another pharmaceutically active agent or compoundselected from the group consisting of a diuretic, cardiac glycoside, ACEinhibitor, angiotensin-2 receptor antagonist, calcium channel blocker,beta blocker, alpha blocker, central alpha agonist, vasodilator, bloodthinner, anti-platelet agent, lipid-lowering agent, and peroxisomeproliferator-activated receptor (PPAR) gamma agonist agent.
 73. Apharmaceutical composition of claim 72 wherein the diuretic is selectedfrom the group consisting of a high ceiling loop diuretic, abenzothiadiazide diuretic, a potassium sparing diuretic, and a osmoticdiuretic.
 74. A pharmaceutical composition of claim 71 wherein thecomposition further comprises another pharmaceutically active agent orcompound selected from the group consisting of an analgesic peptide oragent.
 75. A pharmaceutical composition of claim 74 wherein thecomposition further comprises another pharmaceutically active agent orcompound selected from the group consisting of a laxative agent selectedfrom a bulk-producing agent (e.g. psyllium husk (Metamucil)),methylcellulose (Citrucel), polycarbophil, dietary fiber, apples, stoolsofteners/surfactant (e.g., docusate, Colace, Diocto), a hydrating orosmotic agent (e.g., dibasic sodium phosphate, magnesium citrate,magnesium hydroxide (Milk of magnesia), magnesium sulfate (which isEpsom salt), monobasic sodium phosphate, sodium biphosphate), ahyperosmotic agent (e.g., glycerin suppositories, sorbitol, lactulose,and polyethylene glycol (PEG)).
 76. A method for inhibiting NHE-mediatedantiport of sodium and hydrogen ions, the method comprisingadministering to a mammal in need thereof a pharmaceutically effectiveamount of a compound or pharmaceutical composition of any of claims1-75.
 77. A method for treating a disorder associated with fluidretention or salt overload, the method comprising administering to amammal in need thereof a pharmaceutically effective amount of a compoundor pharmaceutical composition of any of claims 1-75.
 78. A method fortreating a disorder selected from the group consisting of heart failure,chronic kidney disease, end-stage renal disease, liver disease, andperoxisome proliferator-activated receptor (PPAR) gamma agonist-inducedfluid retention, the method comprising administering to a mammal in needthereof a pharmaceutically effective amount of a compound orpharmaceutical composition of any of claims 1-75.
 79. The method ofclaim 78 wherein the heart failure is congestive heart failure.
 80. Amethod for treating hypertension, the method comprising administering toa mammal in need thereof a pharmaceutically effective amount of acompound or pharmaceutical composition of any of claims 1-75.
 81. Amethod of any of claims 77-80 wherein the method comprises administeringa pharmaceutically effective amount of the compound to the mammal inorder to increase the mammal's daily fecal output of sodium and/orfluid.
 82. A method of any of claims 77-81 wherein the method comprisesadministering a pharmaceutically effective amount of the compound to themammal in order to increase the mammal's daily fecal output of sodium byat least about 30 mmol, and/or fluid by at least about 200 ml.
 83. Amethod of any of claims 77-82 wherein the mammal's fecal output ofsodium and/or fluid is increased without introducing another type ofcation in a stoichiometric or near stoichiometric fashion via an ionexchange process.
 84. A method of any of claims 77-83, furthercomprising administering to the mammal a fluid-absorbing polymer toabsorb fecal fluid resulting from the use of the compound that issubstantially active in the gastrointestinal tract to inhibitNHE-mediated antiport of sodium ions and hydrogen ions therein.
 85. Amethod of any of claims 77-84 wherein the compound or composition isadministered to treat hypertension.
 86. A method of any of claims 77-85wherein the compound or composition is administered to treathypertension associated with dietary salt intake.
 87. A method of any ofclaims 77-84 wherein administration of the compound or compositionallows the mammal to intake a more palatable diet.
 88. A method of anyof claims 77-84 wherein the compound or composition is administered totreat fluid overload.
 89. A method of claim 88 wherein the fluidoverload is associated with congestive heart failure.
 90. A method ofclaim 88 wherein the fluid overload is associated with end stage renaldisease.
 91. A method of claim 88 wherein the fluid overload isassociated with peroxisome proliferator-activated receptor (PPAR) gammaagonist therapy.
 92. A method of any of claims 77-84 wherein thecompound or composition is administered to treat sodium overload.
 93. Amethod of any of claim 77-84 wherein the compound or composition isadministered to reduce interdialytic weight gain in ESRD patients.
 94. Amethod of any of claims 77-84 wherein the compound or composition isadministered to treat edema.
 95. A method of any of claim 94 wherein theedema is caused by chemotherapy, pre-menstrual fluid overload orpreeclampsia.
 96. A method of any of claims 77-95 wherein the compoundor composition is administered orally, by rectal suppository, or enema.97. A method of any one of claims 77-96, wherein the method comprisesadministering a pharmaceutically effective amount of the compound orcomposition in combination with one or more additional pharmaceuticallyactive compounds or agents.
 98. A method of claim 97 wherein the one ormore additional pharmaceutically active compounds or agents is selectedfrom the group consisting of a diuretic, cardiac glycoside, ACEinhibitor, angiotensin-2 receptor antagonist, aldosterone antagonist,calcium channel blocker, beta blocker, alpha blocker, central alphaagonist, vasodilator, blood thinner, anti-platelet agent, lipid-loweringagent, and peroxisome proliferator-activated receptor (PPAR) gammaagonist agent.
 99. A method of claim 98 wherein the diuretic is selectedfrom the group consisting of a high ceiling loop diuretic, abenzothiadiazide diuretic, a potassium sparing diuretic, and a osmoticdiuretic.
 100. A method of any of claims 97-99 wherein thepharmaceutically effective amount of the compound or composition, andthe one or more additional pharmaceutically active compounds or agents,are administered as part of a single pharmaceutical preparation.
 101. Amethod of any of claims 97-99 wherein the pharmaceutically effectiveamount of the compound or composition, and the one or more additionalpharmaceutically active compounds or agents, are administered asindividual pharmaceutical preparations.
 102. A method of claim 101wherein the individual pharmaceutical preparation are administeredsequentially.
 103. A method of claim 102 wherein the individualpharmaceutical preparation are administered simultaneously.
 104. Amethod for treating a gastrointestinal tract disorder, the methodcomprising administering to a mammal in need thereof a pharmaceuticallyeffective amount of a compound or pharmaceutical composition of any ofclaims 1-75.
 105. A method of claim 104 wherein the gastrointestinaltract disorder is a gastrointestinal motility disorder.
 106. A method ofclaim 104 wherein the gastrointestinal tract disorder is irritable bowelsyndrome.
 107. A method of claim 104 wherein the gastrointestinal tractdisorder is chronic constipation.
 108. A method of claim 104 wherein thegastrointestinal tract disorder is chronic idiopathic constipation. 109.A method of claim 104 wherein the gastrointestinal tract disorder ischronic constipation occurring in cystic fibrosis patients.
 110. Amethod of claim 104 wherein the gastrointestinal tract disorder isopioid-induced constipation.
 111. A method of claim 104 wherein thegastrointestinal tract disorder is a functional gastrointestinal tractdisorder.
 112. A method of claim 104 wherein the gastrointestinal tractdisorder is selected from the group consisting of chronic intestinalpseudo-obstruction and colonic pseudo-obstruction.
 113. A method ofclaim 104 wherein the gastrointestinal tract disorder is Crohn'sdisease.
 114. A method of claim 104 wherein the gastrointestinal tractdisorder is ulcerative colitis.
 115. A method of claim 104 wherein thegastrointestinal tract disorder is a disease referred to as inflammatorybowel disease.
 116. A method of claim 104 wherein the gastrointestinaltract disorder is associated with chronic kidney disease (stage 4 or 5).117. A method of claim 104 wherein the gastrointestinal tract disorderis constipation induced by calcium supplement.
 118. A method of claim104 wherein the gastrointestinal tract disorder is constipation, andfurther wherein the constipation to be treated is associated with theuse of a therapeutic agent.
 119. A method of claim 104 wherein thegastrointestinal tract disorder is constipation, and further wherein theconstipation to be treated is associated with a neuropathic disorder.120. A method of claim 104 wherein the gastrointestinal tract disorderis constipation, and further wherein the constipation to be treated ispost-surgical constipation (postoperative ileus).
 121. A method of claim104 wherein the gastrointestinal tract disorder is constipation, andfurther wherein the constipation to be treated is idiopathic (functionalconstipation or slow transit constipation).
 122. A method of claim 104wherein the gastrointestinal tract disorder is constipation, and furtherwherein the constipation to be treated is associated with neuropathic,metabolic or an endocrine disorder (e.g., diabetes mellitus, renalfailure, hypothyroidism, hyperthyroidism, hypocalcaemia, MultipleSclerosis, Parkinson's disease, spinal cord lesions, neurofibromatosis,autonomic neuropathy, Chagas disease, Hirschsprung's disease or cysticfibrosis, and the like).
 123. A method of claim 104 wherein thegastrointestinal tract disorder is constipation, and further wherein theconstipation to be treated is due the use of drugs selected fromanalgesics (e.g., opioids), antihypertensives, anticonvulsants,antidepressants, antispasmodics and antipsychotics.
 124. A method fortreating irritable bowel syndrome, the method comprising administeringto a mammal in need thereof a pharmaceutically effective amount of anNHE-3 inhibitor compound or a pharmaceutical composition comprising anNHE-3 inhibitor compound.
 125. A method of claim 124 wherein the NHE-3inhibitor compound or the pharmaceutical composition comprising an NHE-3inhibitor compound is a compound or pharmaceutical composition of any ofclaims 1-75.
 126. A method of any of claims 104-125 wherein the compoundor composition is administered to treat or reduce pain associated with agastrointestinal tract disorder.
 127. A method of any of claims 104-125wherein the compound or composition is administered to treat or reducevisceral hypersensitivity associated with a gastrointestinal tractdisorder.
 128. A method of any of claims 104-125 wherein the compound orcomposition is administered to treat or reduce inflammation of thegastrointestinal tract.
 129. A method of any of claims 104-125 whereinthe compound or composition is administered to reduce gastrointestinaltransit time.
 130. A method of any of claims 104-129 wherein thecompound or composition is administered either orally or by rectalsuppository.
 131. A method of any of claims 104-130 wherein the methodcomprises administering a pharmaceutically effective amount of thecompound or composition, in combination with one or more additionalpharmaceutically active compounds or agents.
 132. A method of claim 131wherein the one or more additional pharmaceutically active agents orcompounds are an analgesic peptide or agent.
 133. A method of claim 131wherein the one or more additional pharmaceutically active agents orcompounds are selected from the group consisting of a laxative agentselected from a bulk-producing agent (e.g. psyllium husk (Metamucil)),methylcellulose (Citrucel), polycarbophil, dietary fiber, apples, stoolsofteners/surfactant (e.g., docusate, Colace, Diocto), a hydrating orosmotic agent (e.g., dibasic sodium phosphate, magnesium citrate,magnesium hydroxide (Milk of magnesia), magnesium sulfate (which isEpsom salt), monobasic sodium phosphate, sodium biphosphate), and ahyperosmotic agent (e.g., glycerin suppositories, sorbitol, lactulose,and polyethylene glycol (PEG)).
 134. A method of any of claims 131-133wherein the pharmaceutically effective amount of the compound orcomposition, and the one or more additional pharmaceutically activecompounds or agents, are administered as part of a single pharmaceuticalpreparation.
 135. A method of any of claims 131-133 wherein thepharmaceutically effective amount of the compound or composition, andthe one or more additional pharmaceutically active compounds or agents,are administered as individual pharmaceutical preparations.
 136. Amethod of claim 135 wherein the individual pharmaceutical preparationare administered sequentially.
 137. A method of claim 135 wherein theindividual pharmaceutical preparation are administered simultaneously.138. A method of treating a disease associated with paracellularpermeability comprising administering an agent which causesintracellular accumulation of protons.
 139. The method of claim 138,wherein the agent is an NHE-3 inhibitor.
 140. The method of claim 138,wherein the NHE-3 inhibitor is a compound of any one of claims 1-47.141. The method of claim 138, wherein the agent is not an NHE-3inhibitor.
 142. The method of any one of claims 138-141, wherein thedisease is selected from gastrointestinal motility disorder, irritablebowel syndrome, chronic constipation, chronic idiopathic constipation,chronic constipation occurring in cystic fibrosis, opioid-inducedconstipation, chronic intestinal pseudo-obstruction, colonicpseudo-obstruction, ulcerative colitis, inflammatory bowel disease,gastrointestinal tract disorder is associated with chronic kidneydisease (stage 4 or 5), constipation induced by calcium supplement,constipation associated with the use of a therapeutic agent,constipation associated with a neuropathic disorder (Parkinson'sdisease, multiple sclerosis), post-surgical constipation (postoperativeileus), idiopathic constipation (functional constipation or slow transitconstipation), constipation associated with neuropathic, metabolic or anendocrine disorder, constipation due the use of drugs selected fromanalgesics (e.g., opioids), antihypertensive, anticonvulsants,antidepressants, antispasmodics and antipsychotics, gastric ulcers,infectious diarrhea, leaky gut syndrome, cystic fibrosisgastrointestinal disease, microscopic colitis, necrotizingenterocolitis, atopy, food allergy, acute inflammation, chronicinflammation, obesity-induced metabolic diseases, kidney disease,chronic kidney disease, diabetic kidney disease, heart disease, heartfailure, congestive heart failure, hypertension, essential hypertension,primary hypertension, salt-sensitive hypertension, liver disease,cirrhosis, nonalcoholic steatohepatitis, nonalcoholic fatty liverdisease, steatosis, primary sclerosing cholangitis, primary biliarycholangitis, portal hypertension, Type 1 diabetes, celiac disease,multiple sclerosis, ankylosing spondylitis, rheumatoid arthritis, lupus,alopecia areata, polymyalgia rheumatica, multiple sclerosis,fibromyalgia, chronic fatigue syndrome, Sjogren's syndrome, vitiligo,thyroiditis, vasculitis, Crohn's disease, ulcerative colitis, urticaria(hives), Raynaud's syndrome, schizophrenia, autism spectrum disorders,multiple sclerosis, hepatic encephalopathy, small intestinal bacterialovergrowth, secondary hyperparathyroidism (PTH), celiac disease,hyperphosphatemia, vascular calcification, elevated FGF-23 levels,cardiac hypertrophy or chronic alcoholism.